HER2/neu-Specific Antibodies and Methods of Using Same

ABSTRACT

This invention relates to antibodies that specifically bind HER2/neu, and particularly chimeric 4D5 antibodies to HER2/neu, which have reduced glycosylation as compared to known 4D5 antibodies. The invention also relates to methods of using the 4D5 antibodies and compositions comprising them in the diagnosis, prognosis and therapy of diseases such as cancer, autoimmune diseases, inflammatory disorders, and infectious disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/994,892 (filed on Jan. 13, 2016), which application is a continuationof U.S. patent application Ser. No. 14/332,239 (filed Jul. 15, 2014;issued as U.S. Pat. No. 9,243,069), which application is a divisional ofU.S. patent application Ser. No. 12/933,885 (filed Jan. 11, 2011; issuedas U.S. Pat. No. 8,802,093), which application is a National StageApplication under 35 U.S.C. §371 of PCT/US2009/038201 (filed Mar. 25,2009), which claims priority to U.S. Patent Application Ser. No.61/041,649 (filed Apr. 2, 2008), which applications are hereinincorporated by reference in their entireties and to which priority isclaimed.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., which are disclosed in both paper andcomputer-readable media, and which paper and computer-readabledisclosures are herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to novel 4D5 antibodies that specifically bindHER2/neu, and particularly novel chimeric 4D5 antibodies to HER2/neu,which have reduced glycosylation and altered effector functions ascompared to known 4D5 antibodies. The invention also relates to methodsof using the antibodies and compositions comprising them in thediagnosis, prognosis and therapy of diseases such as cancer, autoimmunediseases, inflammatory disorders, and infectious disease.

Description of the Related Art

HER2/neu and HER2/neu Receptors

Cellular growth and differentiation processes involve growth factorsthat exert their actions through specific receptors such as the tyrosinekinases. The binding of ligand to a tyrosine kinase receptor triggers acascade of events that eventually lead to cellular proliferation anddifferentiation. (Carpenter et al. (1979) Biochem. 48:193-216; Sachs etal. (1987) Cancer Res. 47:1981-8196). Tyrosine kinase receptors can beclassified into several groups on the basis of sequence similarity anddistinct features. One such family is the ErbB or epidermal growthfactor receptor family, which includes multiple receptors known as HER-1(also known as erbB-1 or EGFR), HER-2 or HER2/neu (also known as erbB-2,c-neu, or p185), HER-3 (also known as erbB-3), and HER-4 (also known aserbB-4). (See, e.g., Carpenter et al., supra; Semba et al. (1985) Proc.Natl. Acad. Sci. (U.S.A.) 82: 6497-6501; Coussens et al. (1985) Science,230:1130-1139, Bargmann et al. (1986) Cell 45:649-657; Kraus et al.(1989) PNAS 86: 9193-9197; Carraway et al. (1994) J. Biol. Chem.269:14303-14306; and Plowman et al. (1993) Nature 366: 473-475; Tzaharet al. (1994) Biol. Chem. 269: 25226-25233).

The ErbB receptors play important roles in propagating signalsregulating cell proliferation, differentiation, motility, and apoptosis,both in normal developmental processes and in human tumorigenesis.(Slamon et al. (1989) Science 244:707-712). For example, the activationof erbB receptors is coupled to and stimulates downstream MAPK-Erk1/2and phosphoinositide-3-kinase (PI₃K)/AKT growth and survival pathways.The deregulation of these pathways in cancer has been linked to diseaseprogression and refractoriness to therapy. (Fukazawa et al. (1996) J.Biol. Chem. 271:14554-14559; Tzahar et al. (1996) Mol. Cell. Biol.16:5276-5287; Lange et al. (1998) J. Biol. Chem. 273:31308-31316;Olayioye et al. (1998) Mol. Cell. Biol. 18:5042-5051; Hackel et al.(1999) Curr. Opin. Cell Biol. 11:184-189). Activation of PI₃K/AKTpromotes cell survival and enhanced tumor aggressiveness, and AKT2 wasreported to be activated and overexpressed in HER2/neu-overexpressingbreast cancers. (Shak (1999) Semin. Oncol. Suppl 12:71-77; Huang et al.(2000) Clinical Cancer Res. 7:2166-2174; Bacus et al. (2002) Oncogene21:3532-3540).

Signaling by the ErbB family of receptors is initiated by ligand bindingwhich triggers homo- or hetero-receptor dimerization, reciprocaltyrosine phosphorylation of the cytoplasmic tails, and activation ofintracellular signal transduction pathways. (Citri et al. (2003) Exp.Cell Res. 284:54). The availability of ligands that bind to and activatethe ErbB receptors is mediated by various metalloproteases, such as theADAM (A Disintegrin And Metalloprotease) family of zinc-dependentmetalloproteases, which catalyze cell surface ectodomain shedding ofspecific proteins. (See Chang and Werb (2001) Trends in Cell Biology11:537-543; Moss and Lambert (2002) Essays in Biochemistry 38:141-153;Seals and Courtneidge (2003) Genes and Development 17:7-30).Specifically, the ADAM family has been shown to cleave ligandsresponsible for activating the ErbB receptors, such as APP and Notch.(Blobel (2005) Nat. Rev. Mol. Cell. Biol. 6:32-43).

An important member of the ErbB family, HER2/neu, is a 185 kDa receptorprotein that was originally identified as the product of thetransforming gene from neuroblastomas of chemically treated rats.HER2/neu has been extensively investigated because of its role inseveral human carcinomas and in mammalian development. (Hynes and Stern(1994) Biochim. et Biophys. Acta 1198:165-184; and Dougall et al. (1994)Oncogene 9:2109-2123; Lee et al. (1995) Nature 378:394-398). The humanHER2/neu gene and HER2/neu protein are described in Semba et al. (1985)Proc. Natl. Acad. Sci. (U.S.A.) 82:6497-6501 and Yamamoto et al. (1986)Nature 319:230-234, and the sequence is available in GenBank asaccession number X03363. HER2/neu comprises four domains: anextracellular domain to which ligand binds; a lipophilic transmembranedomain; a conserved intracellular tyrosine kinase domain; and acarboxyl-terminal signaling domain harboring several tyrosine residuesthat can be phosphorylated. (Plowman et al. (1993) Proc. Natl. Acad.Sci. (U.S.A.) 90:1746-1750). The sequence of the HER2/neu extracellular(ECD) domain was described by Franklin et al. (2004) Cancer Cell.5(4):317-328, and is available in Protein DataBank Record 1S78 (2004).

HER2/neu functions as a growth factor receptor and is often expressed bytumors such as breast cancer, ovarian cancer and lung cancer. HER2/neuis overexpressed in 25-30% of human breast and ovarian cancers, and isassociated with aggressive clinical progression and poor prognosis inthese patients. (Slamon et al. (1987) Science 235:177-182; Slamon et al.(1989) Science 244:707-712). Overexpression of HER2/neu has also beenobserved in other carcinomas including carcinomas of the stomach,endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas andbladder. (See, e.g., King et al. (1985) Science 229:974; McCann et al.(1990) Cancer 65:88-92; Yonemura et al. (1991) Cancer Research 51:1034).

Activation of HER2/neu has been correlated with reduced clinicalresponsiveness to hormone therapy in breast cancer patients. (Wright et.al. (1989) Cancer Res. 49:2087-2090; Kurokawa & Arteaga (2001) Clin.Cancer Res. 7:4436s-42s, 4411s-4412s). Indeed, HER2/neu expression issufficient to convey anti-estrogen resistance. (Benz et. al. (1993)Breast Cancer Res. Treat. 24:85-95). HER2/neu, as well as HER-3, alsoappears to be involved in the onset of hormone resistance in prostatecancer patients. Approximately one-third of prostate cancer patientsreceive hormone therapy treatment aimed at disrupting the action oftesticular and adrenal androgens. As with breast cancer, resistance isinevitable. Recent data suggests that signals emanating from HER2/neuand HER-3 induce a “hormone-refractory” state. (Mellinghoff et. al.(2004) Cancer Cell 6:517-527).

Several truncated and spliced versions of HER2/neu are known. Forexample, a truncated ECD located in the perinuclear cytoplasm of somegastric carcinoma cells is produced by an alternative transcriptgenerated by use of a polyadenylation signal within an intron. (Yamamotoet al. (1986) Nature 319:230-234; and Scott et al. (1993) Mol. Cell.Biol. 13:2247-2257). No particular therapeutic, diagnostic or researchutility has been ascribed to this truncated ECD polypeptide. The ECD ofHER2/neu can also be proteolytically shed from breast carcinoma cells inculture, and is found in the serum of some cancer patients where it ismay be a serum marker of metastatic breast cancer and overall poorprognosis. (Petch et al. (1990) Mol. Cell. Biol. 10:2973-2982; Leitzelet al. (1992) J. Clin. Oncol. 10:1436-1443; Scott et al. (1993) Mol.Cell. Biol. 13:2247-2257; and Lee and Maihle (1998) Oncogene16:3243-3252). In some HER2/neu overexpressing tumor cells,4-aminophenylmercuric acetate (APMA), a well-known metalloproteaseactivator, activates metalloproteases such as ADAM10 and ADAM15 tocleave the HER2/neu receptor into two parts: a truncatedmembrane-associated receptor known as p95, and a soluble ECD known asp105 or ECD105. (See, e.g., Molina et al. (2001) Cancer Res.61:4744-4749; U.S. Patent Publication No. 2004/0247602). Loss of the ECDrenders the p95 receptor a constitutively active tyrosine kinase, whichcan deliver growth and survival signals to cancer cells. (See, e.g.,U.S. Pat. No. 6,541,214).

Studies have shown that in HER2/neu overexpressing breast cancer cells,treatment with antibodies specific to HER2/neu in combination withchemotherapeutic agents (e.g., cisplatin, doxoubicin, taxol) elicits ahigher cytotoxic response than treatment with chemotherapy alone.(Hancock et al. (1991) Cancer Res. 51:4575-4580; Arteaga et al. (1994)Cancer 54:3758-3765; Pietras et al. (1994) Oncogene 9:1829-1838). Onepossible mechanism by which HER2/neu antibodies might enhance responseto chemotherapeutic agents is through the modulation of HER2/neu proteinexpression or by interfering with DNA repair. (Stancovski et al. (1991)Proc. Natl. Acad. Sci. (U.S.A.) 88:8691-8695; Bacus et al. (1992) CellGrowth & Diff. 3:401-411; Bacus et al. (1993) Cancer Res. 53:5251-5261;Klapper et al. (1997) Oncogene 14:2099-2109; Klapper et al. (2000)Cancer Res. 60:3384-3388; Arteaga et al. (2001) J Clinical Oncology19(18s):32s-40s).

A number of monoclonal antibodies and small molecule tyrosine kinaseinhibitors targeting HER-1 or HER2/neu have been developed. For example,a murine monoclonal antibody known as 4D5 recognizes an extracellularepitope (amino acids 529 to 627) in the cysteine-rich II domain ofHER2/neu that resides very close to the transmembrane region. Treatmentof breast cancer cells with 4D5 partially blocks NDF/heregulinactivation of HER2/neu-HER-3 complexes, as measured by receptorphosphorylation assays. (Carter et al. (1992) Proc. Natl. Acad. Sci.(U.S.A.) 89:4285-4289; Sliwkowski et al. (1999) Sem. in Oncol. 26:60-70;Ye et al. (1999) Oncogene 18:731-738; Vogel et al. (2001) Oncology61(suppl 2):37-42; Vogel et al. (2002) Journal of Clinical Oncology20(3):719-726; Fujimoto-Ouchi et al. (2002) Cancer Chemother. Pharmacol.49:211-216). Administration of 4D5 to humans, however, was a clinicalfailure because patients quickly developed HAMA responses, so humanizedforms were developed. The sequence and crystal structure of humanizedantibody 4D5 have been described in U.S. Pat. No. 6,054,297, Carter etal., supra, and Eigenbrot et al. (1993) J. Mol. Biol. 229:969-95.

A humanized form of 4D5 known as trastuzumab (sold as Herceptin® byGenentech, Inc.) was developed and approved for treating cancersinvolving the overexpression or gene amplification of HER2/neu,including breast cancer. (Cobleigh et al. (1999) J. Clin. Oncol.17:2639-2648). Trastuzumab inhibits the APMA-mediated cleavage ofHER2/neu into the ECD and p95 portions in vitro, and is believed to workin vitro through different mechanisms, including the possible inhibitionof HER2/neu shedding. (Pegram et al. (1998) Journal of Clinical Oncology16(8):2659-2671; Baselga et al. (2001) Seminars in Oncology 28(5)(suppl.16):4-11; Baselga et al. (2001) Annals of Oncology 12 (suppl.1):S35-S41). Trastuzumab therapy has various drawbacks, however, such ascardiotoxicity and development of HAHA responses in some patients.

Thus, there is still a need for new or improved forms of HER2/neuantibodies for use in cancer therapies, for example 4D5 antibodieshaving increasing affinity or specificity, reduced potential for HAMA orHAHA responses, altered effector functions, and the like.

Fc Receptors

The interaction of antibody-antigen complexes with cells of the immunesystem results in a wide array of responses, ranging from effectorfunctions such as antibody-dependent cytotoxicity, mast celldegranulation, and phagocytosis to immunomodulatory signals such asregulating lymphocyte proliferation and antibody secretion. All theseinteractions are initiated through the binding of the Fc domain ofantibodies or immune complexes to Fc receptors, which are specializedcell surface receptors on hematopoietic cells. The diversity of cellularresponses triggered by antibodies and immune complexes results from thestructural heterogeneity of Fc receptors. Fc receptors sharestructurally related ligand binding domains which presumably mediateintracellular signaling.

The Fc receptors, members of the immunoglobulin gene superfamily ofproteins, are surface glycoproteins that can bind the Fc portion ofimmunoglobulin molecules. Each member of the family recognizesimmunoglobulins of one or more isotypes through a recognition domain onthe α chain of the Fc receptor. Fc receptors are defined by theirspecificity for immunoglobulin subtypes. Fc receptors for IgG arereferred to as “FcγR,” for IgE as “FεR,” and for IgA as “FcαR.”Different accessory cells bear Fc receptors for antibodies of differentisotype, and the isotype of the antibody determines which accessorycells will be engaged in a given response (Billadeau et al. (2002) J.Clin. Investigat. 2(109):161-81; Gerber et al. (2001) Microbes Infection3:131-139; Ravetch et al. (2001) Annu. Rev. Immunol. 19:275-90; Ravetchet al. (2000) Science 290:84-89; Ravetch (1994) Cell 78(4):553-560;Ravetch et al. (1991) Annu. Rev. Immunol. 9:457-492; see also,Immunobiology: The Immune System in Health and Disease (4th ed. 1999),Elsevier Science Ltd/Garland Publishing, New York). An overview ofvarious receptors is presented in Table 1.

TABLE 1 Receptors for the Fc Regions of Immunoglobulin Isotypes ReceptorBinding Cell Type Effect of Ligation FcγRI IgG1 Macrophages Uptake(CD64) 10⁸ M⁻¹ Neutrophils Stimulation Eosinophils Activation ofrespiratory burst Dendritic cells Induction of killing FcγRII-A IgG1Macrophages Uptake (CD32) 2 × 10⁶ M⁻¹ Neutrophils Granule releaseEosinophils Dendritic cells Platelets Langerhan cells FcγRII-B1 IgG1 Bcells No uptake (CD32) 2 × 10⁶ M⁻¹ Mast cells Inhibition of StimulationFcγRII-B2 IgG1 Macrophages Uptake (CD32) 2 × 10⁶ M⁻¹ NeutrophilsInhibition of Stimulation Eosinophils FcγRIII IgG1 NK cells Induction ofKilling (CD16) 5 × 10⁵ M⁻¹ Eosinophils Macrophages Neutrophils MastCells FcεRI IgE Mast cells Secretion of granules 1010 M⁻¹ EosinophilBasophils FcαRI IgA1, IgA2 Macrophages Uptake (CD89) 10⁷ M⁻¹ NeutrophilsInduction of killing Eosinophils

Each Fcγ receptor (“FcγR”) is an integral membrane glycoprotein,possessing extracellular domains related to a C2-set ofimmunoglobulin-related domains, a single membrane spanning domain and anintracytoplasmic domain of variable length. There are four known FcγRs,designated FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIV. Thereceptors are encoded by distinct genes; however, the extensive homologybetween the family members suggest they arose from a common progenitorperhaps by gene duplication.

Both activating and inhibitory signals are transduced through the FcγRsfollowing ligation. These diametrically opposing functions result fromstructural differences among the different receptor isoforms. Twodistinct domains within the cytoplasmic signaling domains of thereceptor called immunoreceptor tyrosine based activation motifs (ITAMs)or immunoreceptor tyrosine based inhibitory motifs (ITIMS) account forthe different responses. The recruitment of different cytoplasmicenzymes to these structures dictates the outcome of the FcγR-mediatedcellular responses. ITAM-containing FcγR complexes include FcγRI,FcΓRIIA, FcγRIIIA, and FcγRIV, whereas ITIM-containing complexes onlyinclude FcγRIIB.

FcγRI displays high affinity for the antibody constant region andrestricted isotype specificity (Hulett and Hogarth (1994) Adv Immunol57:1-127). FcγRII proteins are 40 KDa integral membrane glycoproteinswhich bind only the complexed IgG due to a low affinity for monomeric Ig(106 M−1). This receptor is the most widely expressed FcγR, present onall hematopoietic cells, including monocytes, macrophages, B cells, NKcells, neutrophils, mast cells, and platelets. FcγRII has only twoimmunoglobulin-like regions in its immunoglobulin binding chain andhence a much lower affinity for IgG than FcγRI. There are three knownhuman FcγRII genes (FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgGin aggregates or immune complexes. Human neutrophils express the FcγRIIAgene. The FcγRIIB gene is expressed on B lymphocytes; its extracellulardomain is 96% identical to FcγRIIA and binds IgG complexes in anindistinguishable manner.

Distinct differences within the cytoplasmic domains of FcγRII-A andFcγRII-B create two functionally heterogenous responses to receptorligation. The FcγRII-A isoform initiates intracellular signaling leadingto cell activation such as phagocytosis and respiratory burst, whereasthe FcγRII-B isoform initiates inhibitory signals, e.g., inhibitingB-cell activation. FcγRIIA clustering via immune complexes or specificantibody cross-linking serves to aggregate ITAMs along withreceptor-associated kinases which facilitate ITAM phosphorylation. ITAMphosphorylation serves as a docking site for Syk kinase, activation ofwhich results in activation of downstream substrates (e.g., PI3K).Cellular activation leads to release of proinflammatory mediators. Whenco-ligated or co-aggregated along with an activating FcγR having anITAM, such as FcγRIIA or FcεRI, the ITIM in FcγRIIB becomesphosphorylated and recruits the SH2 domain of the src homology2-containing inositol phosphatase (SHIP), which in turn isphosphorylated and associates with Shc (Ott (2002) J. Immunol.162(9):4430-4439; Yamanshi et al. (1997) Cell 88:205; Carpino et al.(1997) Cell 88:197). SHIP hydrolyzes phosphoinositol messengers releasedas a consequence of ITAM-containing FcγR-mediated tyrosine kinaseactivation, consequently preventing the influx of intracellular Ca++,and dampening cellular responsiveness to FcγR ligation. Thus, B cellactivation, B cell proliferation and antibody secretion is aborted, andFcγR-mediated phagocytosis is down-regulated (Tridandapani et al. (2002)J. Biol. Chem. 277(7):5082-89).

Specifically, coaggregation of FcγRIIA with FcγRIIB results indown-regulation of phosphorylation of Akt, which is a serine-threoninekinase that is involved in cellular regulation and serves to suppressapoptosis, and coaggregation of FcγRIIB with the high affinity IgEreceptor FcεRI in mast cells leads to inhibition of antigen-induceddegranulation, calcium mobilization, and cytokine production (Long(1999) Annu Rev. Immunol 17:875; Metcalfe et al. (1997) Physiol. Rev.77:1033). Coaggregation of FcγRIIB and the B-cell receptor (BCR) leadsto inhibition of BCR-mediated signaling, and inhibition of cell cycleprogression and cellular survival. Although numerous effector functionsof FcγRIIB-mediated inhibition of BCR signaling are mediated throughSHIP, recently it has been demonstrated that lipopolysaccharide(LPS)-activated B cells from SHIP deficient mice exhibit significantFcγRIIB-mediated inhibition of calcium mobilization, Ins(1,4,5)P3production, and Erk and Akt phosphorylation (Brauweiler et al. (2001)Journal of Immunology 167(1): 204-211).

The size of FcγRIII ranges between 40 and 80 kDa in mouse and man, dueto heterogeneity within this class. Two human genes encode twotranscripts, FcγRIIIA, an integral membrane glycoprotein, and FcγRIIIB,a glycosylphosphatidyl-inositol (GPI)-linked version. One murine geneencodes an FcγRIII homologous to the membrane spanning human FcγRIIIA.The FcγRIII shares structural characteristics with each of the other twoFcγRs. Like FcγRII, FcγRIII binds IgG with low affinity and contains thecorresponding two extracellular Ig-like domains FcγRIIIA is expressed inmacrophages, mast cells, and is the lone FcγR in NK cells. TheGPI-linked FcγRIIIB is currently known to be expressed only in humanneutrophils.

FcγRIV (also known as mFcRIV) requires association of the FcRgamma-chain for optimal expression and function on myeloid cells; itssignaling potential is also enhanced by a cytoplasmic “YEEP” motif thatrecruits the adaptor molecule Crk-L and phosphatidylinositol-3-OHkinase. FcγRIV preferentially binds immunoglobulin E antibodies of the ballotype (IgEb) as well as IgG2a and IgG2b antibodies. Ligation ofFcγRIV by antigen-IgEb immune complexes promotes macrophage-mediatedphagocytosis, presentation of antigen to T cells, production ofproinflammatory cytokines and the late phase of cutaneous allergicreactions (Hirano et al. (2007) Nature Immunology 8:762-771). FcγRIV isa recently identified receptor, conserved in all mammalian species withintermediate affinity and restricted subclass specificity (Nimmerjahn etal. (2005) Immunity 23:41-51; Mechetina et al. (2002) Immunogenetics54:463-468; Davis et al. (2002) Immunol Rev 190:23-36). FcRIII and FcRIVare physiologically important activation FcRs for mediating inflammatorydisease triggered by cytotoxic antibodies or pathogenic immunecomplexes. FcRIV is found on dendritic cells, macrophages, monocytes andneutrophils.

Despite all such advances, a need remains for anti-HER2/neu antibodiesthat possess therapeutic use in the treatment of autoimmunity, cancer,inflammatory disease, and/or transplantation, and exhibit improvedability to mediate effector function from the Fc receptors. The presentinvention is directed to this and other needs.

SUMMARY OF THE INVENTION

Embodiments of the invention provide various polypeptides, for example apolypeptide comprising an immunoglobulin light chain variable domainhaving the amino acid sequence of SEQ ID NO: 4, a polypeptide comprisingan immunoglobulin light chain having the amino acid sequence of SEQ IDNO: 2, and a polypeptide comprising a chimeric 4D5 immunoglobulin lightchain comprising an N65S substitution. Other embodiments provide apolypeptide comprising an immunoglobulin heavy chain having the aminoacid sequence of SEQ ID NO: 7, a polypeptide comprising animmunoglobulin heavy chain having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13,and a polypeptide comprising a 4D5 immunoglobulin heavy chain comprisingvarious substitutions.

The polypeptides may be antibodies, and may specifically bind humanHER2/neu. The polypeptides and antibodies may comprise a variant Fcdomain, which comprises one or more modifications, which may confer aphenotype alteration on the polypeptide or antibody, including alteredeffector function, increased or decreased binding to an FcγR, etc. Theembodiments of the invention also provide polynucleotides encoding thepolypeptides and antibodies, vectors comprising the polynucleotides, andhost cells comprising the vectors. Methods of producing the polypeptidesand antibodies, as well as methods of treating various diseases anddisorders, are also provided.

In detail, the invention provides a polypeptide comprising a chimeric4D5 immunoglobulin light chain comprising an N65S substitution, andparticularly the embodiments of such polypeptide wherein the polypeptidecomprises a light chain variable domain having the amino acid sequenceof SEQ ID NO: 4 or of SEQ ID NO: 2.

The invention particularly concerns the embodiments of such polypeptideswherein the polypeptide is an antibody, and more particularly, anantibody that comprises a variant Fc domain having at least onemodification in the Fc domain. The invention particularly concernsembodiments wherein the modification comprises at least one substitutionselected from the group consisting of L235V, F243L, R292P, Y300L, V305I,and P396L.

The invention further concerns the embodiments of such antibodieswherein the modification in the Fc domain comprises:

-   -   (A) at least one substitution selected from the group consisting        of F243L, D270E, R292P, S298N, Y300L, V305I, A330V, and P396L;    -   (B) at least two substitutions selected from the group        consisting of F243L and P396L; F243L and R292P; and R292P and        V305I;    -   (C) at least three substitutions selected from the group        consisting of F243L, R292P and Y300L; F243L, R292P and V305I;        F243L, R292P and P396L; and R292P, V305I and P396L; or    -   (D) at least four substitutions selected from the group        consisting of F243L, R292P, Y300L and P396L; and F243L, R292P,        V305I and P396L;        and more particularly concerns antibodies wherein the        modification in the Fc domain comprises:    -   (1) F243L, R292P, and Y300L;    -   (2) L235V, F243L, R292P, Y300L, and P396L; or    -   (3) F243L, R292P, Y300L, V305I, and P396L.

The invention further concerns the embodiments of such antibodieswherein the variant Fc domain exhibits, as compared to a wild-type Fcdomain:

-   -   (A) enhanced antibody dependent cell mediated cytotoxicity        (ADCC);    -   (B) increased binding to FcγRIIA or to FcγRIIIA;    -   (C) decreased binding to FcγRIIB; or    -   (D) increased binding to FcγRIIB.

The invention further concerns a polypeptide comprising animmunoglobulin heavy chain comprising a variable domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 9, SEQ IDNO: 11, and SEQ ID NO: 13.

The invention further concerns a polypeptide comprising a 4D5immunoglobulin heavy chain comprising:

-   -   (A) at least one substitution selected from the group consisting        of: F243L, D270E, R292P, S298N, Y300L, V305I, A330V and P396L;    -   (B) at least two substitutions selected from the group        consisting of:        -   (1) F243L and P396L;        -   (2) F243L and R292P; and        -   (3) R292P and V305I;    -   (C) at least three substitutions selected from the group        consisting of:        -   (1) F243L, R292P and Y300L;        -   (2) F243L, R292P and V305I;        -   (3) F243L, R292P and P396L; and        -   (4) R292P, V3051 and P396L;    -   (D) at least four substitutions selected from the group        consisting of:    -   (1) F243L, R292P, Y300L and P396L; and    -   (2) F243L, R292P, V305I and P396L;        or    -   (E) at least F243L, R292P, Y300L, V305I and P396 substitutions.

The invention further concerns the embodiment of such a polypeptidewherein the polypeptide is an antibody.

The invention further concerns the embodiment of the above-describedantibodies, wherein the antibody further comprises an immunoglobulinheavy chain comprising a variable domain having the amino acid sequenceof SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.

The invention further concerns the embodiment of the above-describedantibodies, wherein the antibody comprises:

-   -   (A) at least one substitution selected from the group consisting        of: F243L, D270E, R292P, S298N, Y300L, V305I, A330V and P396L;    -   (B) at least two substitutions selected from the group        consisting of:        -   (1) F243L and P396L;        -   (2) F243L and R292P; and        -   (3) R292P and V305I;    -   (C) at least three substitutions selected from the group        consisting of:        -   (1) F243L, R292P and Y300L;        -   (2) F243L, R292P and V305I;        -   (3) F243L, R292P and P396L; and        -   (4) R292P, V305I and P396L;    -   (D) at least four substitutions selected from the group        consisting of:        -   (1) F243L, R292P, Y300L and P396L; and        -   (2) F243L, R292P, V305I and P396L;            or    -   (E) at least F243L, R292P, Y300L, V305I and P396 substitutions.

The invention further concerns the embodiments of the above-describedantibodies wherein the immunoglobulin light chain comprises a variabledomain having the amino acid sequence of SEQ ID NO: 4 and theimmunoglobulin heavy chain comprises an amino acid sequence of SEQ IDNO: 9, SEQ ID NO: 11 or SEQ ID NO:13.

The invention further concerns the embodiments of the above-describedantibodies wherein the antibody is a F(ab′)₂ fragment, a F(ab) fragment,a single chain antibody, a monoclonal antibody or a diabody.

The invention further concerns the use of the above-described antibodiesin the manufacture of a medicament for the treatment of cancer in apatient, and particularly, wherein the cancer is a HER2/neu-expressingcancer and wherein the antibody binds human HER2/neu.

The invention further concerns a method of treating cancer whichcomprises providing to a patient in need thereof an effective amount ofthe above-described antibodies, and particularly, wherein the cancer isa HER2/neu-expressing cancer and wherein the antibody binds humanHER2/neu.

The invention further concerns the use of such antibodies wherein thetreatment further comprises the step of administering a secondtherapeutic agent simultaneously or sequentially with the antibody,wherein the second therapeutic agent is selected from the groupconsisting of an anti-angiogenic agent, an anti-neoplastic agent, achemotherapeutic agent, and a cytotoxic agent.

Additional advantages and features of the present invention will beapparent from the following detailed description, drawings and examples,which illustrate preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a sequence alignment comparing the sequences of the lightchain variable region of a chimeric 4D5 antibody of a preferredembodiment (SEQ ID NO: 4) with murine (SEQ ID NO: 3) and humanized (SEQID NO: 5) 4D5 antibodies.

FIG. 2 depicts a comparison between the sequences of the heavy chains ofch4D5-wild-type Fc (“WT”) (SEQ ID NO: 7), ch4D5-FcMT1 (“MT1”) (SEQ IDNO: 9), ch4D5-FcMT2 (“MT2”) (SEQ ID NO: 11), and ch4D5-FcMT3 (“MT3”)(SEQ ID NO: 13). The CDRs are indicated with black bars shown underneaththe pertinent residues.

FIG. 3 depicts a BIACore analysis of ch4D5-wild-type Fc (panel A), ch4D5(panel B) and trastuzumab (panel C) binding.

FIG. 4 depicts the effect of ch4D5 on the proliferation of SKBR3 cellsin vitro.

FIG. 5 depicts the enhanced anti-tumor activity of various antibodies ofthe present embodiments in non-transgenic mice.

FIG. 6 depicts the enhanced anti-tumor activity of various antibodies ofthe present embodiments in hCD16A transgenic mice.

FIG. 7 depicts the role of mFcRIV and hCD16A in tumor growth inhibitionby various antibodies of the present embodiments in non-transgenic andtransgenic mice.

FIG. 8 depicts the enhanced anti-tumor activity of various antibodies ofthe present embodiments in hCD16A transgenic mice.

FIG. 9 illustrates representative immunohistochemical staining of cellsfrom various cancer cell lines for HER2/neu. The various panelsrepresent the different cell lines, i.e., Panel A: MDA-MB-435; Panel B:MDA-MB-231; Panel C: A549; Panel D: OVCAR-8; Panel E: MCF-7; Panel F:BT-20; Panel G: HT-29; Panel H: ZR75-1; Panel I: JIMT-1; Panel J:MDA-MB-453; Panel K: BT-474; Panel L: SKBR-3; and Panel M: mSKOV-3.

FIG. 10 depicts the results of ADCC assays performed to test the abilityof various ch4D5 antibodies of the present embodiments to mediate ADCCin cancer cell lines (MDA-MB-435 in Panel A; MDA-MB-231 in Panel B)having very low or no HER2/neu expression levels (DAKO score of 0).

FIG. 11 depicts the results of ADCC assays performed to test the abilityof various ch4D5 antibodies of the present embodiments to mediate ADCCin cancer cell lines (A549 in Panel A; OVCAR-8 in Panel B; MCF-7 inPanel C; BT-20 in Panel D; HT-29 in Panel E) having low HER2/neuexpression levels (DAKO score of 1+).

FIG. 12 depicts the results of ADCC assays performed to test the abilityof various ch4D5 antibodies of the present embodiments to mediate ADCCin cancer cell lines (ZR75-1 in Panel A; JIMT-1 in Panel B) havingmoderate HER2/neu expression levels (DAKO score of 2+).

FIG. 13 depicts the results of ADCC assays performed to test the abilityof various ch4D5 antibodies of the present embodiments to mediate ADCCin cancer cell lines (MDA-MB-453 in Panel A; BT-474 in Panel B; SKBR-3in Panel C; mSKOV-3 in Panel D) having high HER2/neu expression levels(DAKO score of 3+).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel antibodies and methods fortreatment, diagnosis and prognosis for certain cancers using antibodiesagainst HER2/neu. In particular, the present invention providesanti-HER2/neu antibodies that are particularly useful as selectivecytotoxic agents for HER2/neu overexpressing cells, for example chimeric4D5 antibodies to HER2/neu, which have reduced glycosylation and alteredeffector functions as compared to known 4D5 antibodies. The inventionalso provides methods of using the antibodies and compositionscomprising them in the diagnosis, prognosis and therapy of diseases suchas cancer, autoimmune diseases, inflammatory disorders, and infectiousdisease.

Reference will now be made in detail to the presently preferredembodiments of the invention, which, together with the drawings and thefollowing examples, serve to explain the principles of the invention.These embodiments are described in sufficient detail to enable thoseskilled in the art to practice the invention, and it is to be understoodthat other embodiments may be utilized, and that structural, biological,and chemical changes may be made without departing from the spirit andscope of the present invention. Unless otherwise defined, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. One skilled in the art may refer to general reference texts forsuch definitions or for detailed descriptions of techniques discussedherein. These texts include Current Protocols in Molecular Biology(Ausubel et al., eds., John Wiley & Sons, and supplements through March2008), Molecular Cloning: A Laboratory Manual (Sambrook and Russell, 3rded., 2001); Single-Molecule Techniques: A Laboratory Manual (Selvin &Ha, eds., Cold Spring Harbor Press, 2008); Current Protocols in NucleicAcid Chemistry (Beaucage et al., eds., John Wiley & Sons, Inc., 2000);Current Protocols in Immunology (Coligan et al., eds., John Wiley &Sons, N.Y., and supplements through March 2008), Making and UsingAntibodies: A Practical Handbook (Howard & Kaser, eds., CRC, 2006);Using Antibodies: A Laboratory Manual (Harlow & Lane, Cold Spring HarborPress, 1999); Binding and Kinetics for Molecular Biologists (Goodrich &Kugel, Cold Spring Harbor Press, 2007); Current Protocols inPharmacology (Enna et al., eds., John Wiley & Sons, N.Y., andsupplements through March 2008), The Pharmacological Basis ofTherapeutics (Goodman & Gilman, 11th ed., 2006), and Remington: TheScience and Practice of Pharmacy (Lippincott Williams & Wilkins, 21stedition (2005), for example

A. Definitions

As used herein, the term “ADCC” refers to Antibody Dependent CellularCytotoxicity, an in vitro cell-mediated reaction in which nonspecificcytotoxic cells that express FcγRs (e.g., monocytic cells such asNatural Killer (NK) cells and macrophages) recognize bound antibody on atarget cell and subsequently cause lysis of the target cell.

As used herein, the term “antibody” refers to monoclonal antibodies,multispecific antibodies, human antibodies, humanized antibodies,synthetic antibodies, chimeric antibodies, polyclonal antibodies,camelized antibodies, single-chain Fvs (scFv), single chain antibodies,immunologically active antibody fragments (e.g., antibody fragmentscapable of binding to an epitope, e.g., Fab fragments, Fab′ fragments,F(ab′)₂ fragments, Fv fragments, fragments containing either a VL or VHdomain or a complementary determining region (CDR) thatimmunospecifically binds an antigen, etc.), bi-functional ormulti-functional antibodies, disulfide-linked bispecific Fvs (sdFv),intrabodies, and diabodies, and epitope-binding fragments of any of theabove. In particular, the term antibodies is intended to encompassimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass (see, e.g., United States Patent Publication Nos.:20040185045; 20050037000; 20050064514; 20050215767; 20070004909;20070036799; 20070077246; and 20070244303).

Reference to a “B cell antigen receptor” or “BCR” is intended toreference the B cell antigen receptor, which includes a membraneimmunoglobulin (mlg) antigen binding component, or a biologically activeportion thereof (i.e, a portion capable of binding a ligand and/orcapable of associating with a transducer component), and transducerCD79a and CD79b components, or biologically active portions thereof(i.e., a portion capable of transducing an intracellular signal and/orcapable of associating with an extracellular ligand binding portion).

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. As used herein,cancer explicitly includes, leukemias and lymphomas. In someembodiments, cancer refers to a benign tumor, which has remainedlocalized. In other embodiments, cancer refers to a malignant tumor,which has invaded and destroyed neighboring body structures and spreadto distant sites. In some embodiments, the cancer is associated with aspecific cancer antigen.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

The term “chimeric,” when referring to antibodies, refers to an antibodyin which a portion of a heavy and/or light chain is identical to orhomologous with an antibody from one species (e.g., mouse) or antibodyclass or subclass, while the remaining portion is identical to orhomologous with an antibody of another species (e.g., human) or antibodyclass or subclass, so long as they exhibit the desired biologicalactivity. Chimeric antibodies of interest herein include “primatized”antibodies comprising variable domain antigen-binding sequences derivedfrom a non-human primate (e.g., Old World Monkey, Ape, etc.) and humanconstant region sequences.

As used herein, the term “Complementarity Determining Region” or “CDR”refers to the amino acid residues of an antibody variable domain thatare necessary for antigen binding. Each variable domain typically hasthree CDR regions identified as CDR₁, CDR₂ and CDR₃.

As used herein, the term “diabody molecule” refers to a complex of twoor more polypeptide chains or proteins, each comprising at least oneV_(L) and one V_(H) domain or fragment thereof, wherein both domains arecomprised within a single polypeptide chain In certain embodiments a“diabody molecule” includes molecules comprising an Fc or a hinge-Fcdomain. Said polypeptide chains in the complex may be the same ordifferent, i.e., the diabody molecule may be a homo-multimer or ahetero-multimer. In specific aspects, a “diabody molecule” includesdimers or tetramers or said polypeptide chains containing both a V_(L)and V_(H) domain. The individual polypeptide chains comprising themultimeric proteins may be covalently joined to at least one otherpeptide of the multimer by interchain disulfide bonds.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject. In particular, theterm “autoimmune disease” is used interchangeably with the term“autoimmune disorder” to refer to a condition in a subject characterizedby cellular, tissue and/or organ injury caused by an immunologicreaction of the subject to its own cells, tissues and/or organs. Theterm “inflammatory disease” is used interchangeably with the term“inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Thus, certain disorders may be characterized as bothautoimmune and inflammatory disorders.

The term “effector cell” as used herein refers to a cell of the immunesystem that expresses one or more Fc receptors and mediates one or moreeffector functions. Effector cells include but are not limited tomonocytes, macrophages, neutrophils, dendritic cells, eosinophils, mastcells, platelets, B cells, large granular lymphocytes, Langerhans'cells, natural killer (NK) cells, and may be from any organism includingbut not limited to humans, mice, rats, rabbits, and monkeys.

The term “effector cell” refers to biological activities attributable tothe interaction of an antibody Fc region with an Fc receptor or ligand.An antibody may have one or more effector functions. Non-limitingexamples of antibody effector functions include antibody-dependentcell-mediated cytotoxicity (ADCC), C1q binding, complement dependentcytotoxicity (CDC), down regulation of cell surface receptors (e.g.,B-cell receptor; BCR), opsonization, opsonophagocytosis, cell binding,and rosetting. Effector functions include both those that operate afterthe binding of an antigen and those that operate independent of antigenbinding.

As used herein, the term “epitope” refers to that portion of apolypeptide or protein or a non-protein molecule that isimmunospecifically bound by an antibody. An epitope may have immunogenicactivity, such that it elicits an antibody production response in ananimal. The ability of an epitope to immunospecifically bind an antibodymay be determined by for example, an immunoassay. Epitopes need notnecessarily be immunogenic.

The terms “Fc receptor” or “FcR” are used herein to describe a receptorthat binds to the Fc region of an antibody. An exemplary FcR is a nativesequence human FcR. An FcR may be one which binds an IgG antibody (agamma receptor) and includes receptors of the FcγRI, FcγRII, FcγRIII,and FcγRIV subclasses, including allelic variants and alternativelyspliced forms of these receptors, e.g., there are at least two knownFcγRII receptors, FcγRIIA and FcγRIIB. The term FcR also includes theneonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus.

As used herein, the term “Fc region” is used to define a C-terminalregion of an IgG heavy chain. Although the boundaries may vary slightly,the human IgG heavy chain Fc region is defined to stretch from Cys226 tothe carboxy terminus. The Fc region of an IgG comprises two constantdomains, C_(H2) and C_(H3). The C_(H2) domain of a human IgG Fc region(also referred to as “Cγ2” domain) usually extends from amino acid 231to amino acid 338, and the C_(H3) domain of a human IgG Fc regionusually extends from amino acids 342 to 447.

The term “glycosylation site” refers to an amino acid residue orresidues that is recognized by a mammalian cell as a location for theattachment of sugar residues. Amino acid residues to whichcarbohydrates, such as oligosaccharides, are attached are usuallyasparagine (N-linkage), serine (O-linkage), and threonine (O-linkage)residues. The specific sites of attachment usually have a characteristicsequence of amino acids, referred to as a “glycosylation site sequence.”The glycosylation site sequence for N-linked glycosylation is:Asn-X-Ser/Thr, where X can be any of the conventional amino acids otherthan proline. The Fc region of human IgG has two N-linked glycosylationsites, one in each of the C_(H2) domains, at the asparagine at position297 (Asn 297).

As used herein, the term “HAMA response” refers to the Human Anti-MouseAntibody response, which is a deleterious immunogenic response thatoccurs when a human immune system recognizes a murine antibody asforeign and attacks it. A HAMA response can cause toxic shock or death.Chimeric and humanized antibodies reduce the likelihood of a HAMAresponse by decreasing the non-human portions of administeredantibodies, but there is still potential for a Human Anti-Human Antibodyresponse (“HAHA response”) immune response to such antibodies.

The terms “heavy chain,” “light chain” (“CL”), “light chain variableregion” (“VL”), “heavy chain variable region” (“VH”), “framework region”(“FR”), “heavy chain constant domain (“CH”), “light chain constantdomain (“CL”) refer to domains in naturally occurring immunoglobulinsand the corresponding domains of synthetic (e.g., recombinant) bindingproteins (e.g., humanized antibodies). The basic structural unit ofnaturally occurring immunoglobulins (e.g., IgG) is a tetramer having twolight chains and two heavy chains. Usually naturally occurringimmunoglobulin is expressed as a glycoprotein of about 150 KDa, althoughIgG can also be produced in a non-glycosylated form. The amino-terminal(“N”) portion of each chain includes a variable region of about 100 to110 or more amino acids primarily responsible for antigen recognition.The carboxy-terminal (“C”) portion of each chain defines a constantregion, with light chains having a single constant domain and heavychains usually having three constant domains and a hinge region. Thus,the structure of the light chains of a naturally occurring IgG moleculeis N-VL-CL-C and the structure of IgG heavy chains isN-VH-CH1-H-CH2-CH3-C (where H is the hinge region). The variable regionsof an IgG molecule consists of the complementarity determining regions(CDRs), which contain the residues in contact with antigen and non-CDRsegments, referred to as framework segments, which maintain thestructure and determine the positioning of the CDR loops. Thus, the VLand VH domains have the structure N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.

As used herein, the term “heterologous” nucleic acid denotes DNA, RNA,etc. that is introduced into a host cell. The nucleic acid may bederived from any of a variety of sources including genomic DNA, mRNA,cDNA, synthetic DNA and fusions or combinations of these. The nucleicacid may include a polynucelotide from the same cell or cell type as thehost or recipient cell or a polynucleotide from a different cell type,for example, from a mammal or plant, and may, optionally, include markeror selection genes, for example, antibiotic resistance genes,temperature resistance genes, etc.

The term “hinge region” is generally defined as stretching from Glu216to Pro230 of human IgG1. Hinge regions of other IgG isotypes may bealigned with the IgG1 sequence by placing the first and last cysteineresidues forming inter-heavy chain S—S bonds in the same positions.

As used herein, the term “humanized” has its usual meaning in the art.In general terms, humanization of a non-human antibody involvessubstituting the CDR sequences from non-human immunoglobulin V_(L) andV_(H) regions into human framework regions. Further, as used herein,“humanized” antibodies may comprise additional substitutions andmutations in the CDR and/or framework regions introduced to increaseaffinity or for other purposes. For example, substitution of nonhumanframework residues in the human sequence can increase affinity. Theresulting variable domains have non-human CDR sequences and frameworksequences derived from human antibody framework sequence(s) or a humanconsensus sequence. A variety of different human framework regions maybe used singly or in combination as a basis for a humanized antibody.

As used herein, the term “immunomodulatory agent” and variations thereofrefer to an agent that modulates a host's immune system. In certainembodiments, an immunomodulatory agent is an immunosuppressant agent. Incertain other embodiments, an immunomodulatory agent is animmunostimulatory agent. Immunomodatory agents include, but are notlimited to, small molecules, peptides, polypeptides, fusion proteins,antibodies, inorganic molecules, mimetic agents, and organic molecules.

As used herein, the term “immunospecifically binds,” refers to thespecific binding exhibited between an antibody and the epitope that itrecognizes. Such binding will typically exhibit a K_(D) of at leastabout 0.1 mM, more usually at least about 1 μM, preferably at leastabout 0.1 μM or less, and most preferably, 0.01 μM or less. Preferably,the antibodies of the invention immunospecifically bind to proteins withhigh affinity (e.g., low K_(D)).

An antibody that immunospecifically binds to an antigen may bind toother peptides or polypeptides with lower affinity as determined by,e.g., immunoassays, BIAcore, or other assays known in the art.Preferably, molecules that specifically bind an antigen do not crossreact with other proteins. Molecules that specifically bind an antigencan be identified, for example, by immunoassays, BIAcore, or othertechniques known to those of skill in the art.

The term “Antibody Engineering Technology Art” as used herein refers totechnology disclosed in U.S. Provisional Patent Application Nos.60/781,564; 60/945,523; 61/015,106; filed Dec. 19, 2007, and 61/019,051filed Jan. 4, 2008; US 20040185045; US 20040197347; US 20040197866; US20050037000; US 20050064514; US 20050215767; US 20060134709; US20060177439; US 20070004909; US 20070036799; US 20070037216; US20070077246; US 20070244303; US 20080044429; US 20080050371; 11/869,410;11/952,568; U.S. Pat. No. 7,112,439; WO 04/063351; WO 06/088494; WO07/024249; WO 06/113665; WO 07/021841; WO 07/106707; or PCT/US07/86793

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts, and the term “polyclonal antibody” as used herein refersto an antibody obtained from a population of heterogenous antibodies.Monoclonal antibodies are highly specific, being directed against asingle epitope. In addition to their specificity, monoclonal antibodiesare advantageous in that they may be synthesized without contaminationby other antibodies. The term “monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method.

As used herein, the terms “nucleic acids” and “nucleotide sequences”include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g.,mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNAmolecules, and analogs of DNA or RNA molecules. Such analogs can begenerated using, for example, nucleotide analogs, which include, but arenot limited to, inosine or tritylated bases. Such analogs can alsocomprise DNA or RNA molecules comprising modified backbones that lendbeneficial attributes to the molecules such as, for example, nucleaseresistance or an increased ability to cross cellular membranes. Thenucleic acids or nucleotide sequences can be single-stranded,double-stranded, may contain both single-stranded and double-strandedportions, and may contain triple-stranded portions, but preferably isdouble-stranded DNA.

“Substantial sequence identity,” as used herein, refers to two or moresequences or subsequences (e.g., domains) that have at least about 80%amino acid residue identity, preferably at least about 90%, or at leastabout 95% identity when compared and aligned for maximum correspondence.Sequence identity between two similar sequences (e.g., antibody variableregions) can be measured by algorithms such as that of Smith & Waterman,1981, Adv. Appl. Math. 2:482 [local homology algorithm], Needleman &Wunsch, 1970, J. Mol. Biol. 48:443 [homology alignment algorithm],Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 [searchfor similarity method], or Altschul et al., 1990, J. Mol. Biol.215:403-10 [BLAST algorithm]. When using any of the aforementionedalgorithms, the default parameters (for Window length, gap penalty,etc.) are used. An amino acid sequence is said to be “substantiallysimilar to” a second sequence when the degree of sequence identity is atleast about 70% identical, preferably at least about 80%, or at leastabout 90%, or even at least about 95%, identical. A nucleic acidsequence is said to be “substantially similar to” a second sequence wheneither: (1) the degree of sequence identity is at least about 70%identical, preferably at least about 80%, or at least about 90%, or evenat least about 95%, identical, or the nucleic acid sequence encodes apolypeptide that is at least about 70% identical, preferably at leastabout 80%, or at least about 90%, or even at least about 95%, identicalto the polypeptide encoded by the second sequence. Sequences that aresubstantially identical are also substantially similar.

When referring to antibodies, the assignment of amino acids to eachdomain is in accordance with Kabat, Sequences Of Proteins OfImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991), which is expressly incorporated herein by reference.Throughout the present specification, the numbering of the residues inan IgG heavy chain is that of the EU index as in Kabat, and refers tothe numbering of the human IgG1 EU antibody.

B. Antibodies

The present invention particularly encompasses chimeric antibodies andpolypeptides that specifically bind to HER2/neu, preferably humanHER2/neu. The antibodies have reduced glycosylation as compared to known4D5 antibodies such as murine 4D5 and trastuzumab, due to the removal ofa glycosylation site in the variable region of the light chain. Inparticular, the antibodies lack a glycosylation site in the variableregion of the light chain, which in the native murine 4D5 comprises anN-R-S sequence at positions 65, 66 and 67. Preferably the antibodieshave enhanced binding affinity for HER2/neu, and more preferably theantibodies have enhanced effector function, both as compared to a native4D5 antibody.

The antibodies comprise a chimeric 4D5 immunoglobulin light chainlacking an N-linked glycosylation site at positions 65, 66 and 67 of thelight chain variable region. In a particular embodiment, the antibodiescomprise a modification, preferably a substitution, at position 65 ofthe variable region of the light chain (V_(L)). In a preferredembodiment, the antibodies comprise a light chain encoded by the nucleicacid sequence of SEQ ID NO: 1, or comprise the amino acid sequence ofSEQ ID NO: 2. The nucleic acid and amino acid sequences of a preferredlight chain of the present invention are presented below:

Chimeric light chain nucleic acid sequence(SEQ ID NO: 1):gacatcgtga tgacccagtc ccacaagttc atgtccacct ctgtgggcga tagggtcagc  60atcacctgca aggccagcca ggatgtgaat actgctgtag cctggtatca gcagaaacca 120ggacattctc ccaaactgct gatttactcc gcatccttcc ggtacactgg agtccctgat 180cgcttcactg gcagcagatc tgggacagat ttcactttca ccatcagcag tgtgcaggct 240gaagacctgg cagtttatta ctgtcagcaa cattatacta cacctcccac cttcggaggg 300ggtaccaagg tggagatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645Chimeric light chain amino acid sequence (SEQ ID NO: 2):DIVMTQSHKF MSTSVGDRVS ITCKASQDVN TAVAWYQQKP GHSPKLLIYS ASFRYTGVPD  60RFTGSRSGTD FTFTISSVQA EDLAVYYCQQ HYTTPPTFGG GTKVEIKRTV AAPSVFIFPP 120SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214

These antibodies having a modification at positions 65 of the V_(L)region lack an N-linked glycosylation site found in the native murine4D5 antibody, as can be seen in FIG. 1, which depicts an exemplarycomparison between the V_(L) region amino acid sequences of a chimeric4D5 antibody having an N65S modification (SEQ ID NO: 4), and the nativemurine (SEQ ID NO: 3) and humanized (SEQ ID NO: 5) 4D5 antibodies. Inanother preferred embodiment, the antibodies comprise a N65Smodification in the V_(L) region, and preferably have a V_(L) regionamino acid sequence of SEQ ID NO: 4. These sequences are presentedbelow:

Native murine V_(L) region amino acid sequence (SEQ ID NO: 3):DIVMTQSHKF MSTSVGDRVS ITCKASQDVN TAVAWYQQKP GHSPKLLIYS ASFRYTGVPD  60RFTGNRSGTD FTFTISSVQA EDLAVYYCQQ HYTTPPTFGG GTKLEIKRA 109Chimeric V_(L) region amino acid sequence (SEQ ID NO: 4):DIVMTQSHKF MSTSVGDRVS ITCKASQDVN TAVAWYQQKP GHSPKLLIYS ASFRYTGVPD  60RFTGSRSGTD FTFTISSVQA EDLAVYYCQQ HYTTPPTFGG GTKVEIKRT 109Humanized V_(L) region amino acid sequence (SEQ ID NO: 5):DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLESGVPS  60RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRT 109

In one embodiment, the heavy chain has a murine 4D5 wild-type Fc region,preferably encoded by the nucleic acid sequence of SEQ ID NO: 6, orhaving the amino acid sequence of SEQ ID NO: 7. These sequences arepresented below:

Heavy chain having wild-type Fc region nucleic acid sequence (SEQ ID NO: 6):caggttcagc tgcagcagtc tggccctgag ctggtgaagc caggggcctc actcaagttg   60tcctgtacag cttctggctt caacatcaaa gacacctata tccactgggt gaaacagagg  120cctgaacagg gcctggaatg gattggaagg atttatccta ccaatggcta tactagatat  180gacccaaagt tccaggacaa ggccactatc acagcagaca catcctccaa cacagcctac  240ctgcaagtca gccgcctgac atctgaggac actgccgtct attactgctc ccggtgggga  300ggggacggct tctatgctat ggactactgg ggtcagggag cctccgtgac cgtgagctcc  360gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg  420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg  480tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca  540ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc  600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc  660aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga  720ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct  780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg  840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac  900agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag  960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc tccgggtaaa tga 1353Heavy chain having wild-type Fc region amino acid sequence (SEQ ID NO: 7):QVQLQQSGPE LVKPGASLKL SCTASGFNIK DTYIHWVKQR PEQGLEWIGR IYPTNGYTRY  60DPKFQDKATI TADTSSNTAY LQVSRLTSED TAVYYCSRWG GDGFYAMDYW GQGASVTVSS 120ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 180GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG 240PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 360LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420QQGNVFSCSV MHEALHNHYT QKSLSLSPGK  450

In other embodiments, the heavy chain is a variant of a murine 4D5antibody heavy chain, preferably comprising a variant Fc region, andmore preferably comprising a variant Fc region such as FcMT1, FcMT2, orFcMT3. These sequences are presented below:

Heavy chain having FcMT1 variant Fc region nucleic acid sequence(SEQ ID NO: 8):caggttcagc tgcagcagtc tggccctgag ctggtgaagc caggggcctc actcaagttg   60tcctgtacag cttctggctt caacatcaaa gacacctata tccactgggt gaaacagagg  120cctgaacagg gcctggaatg gattggaagg atttatccta ccaatggcta tactagatat  180gacccaaagt tccaggacaa ggccactatc acagcagaca catcctccaa cacagcctac  240ctgcaagtca gccgcctgac atctgaggac actgccgtct attactgctc ccggtgggga  300ggggacggct tctatgctat ggactactgg ggtcagggag cctccgtgac cgtgagctcc  360gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg  420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg  480tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca  540ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc  600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc  660aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga  720ccgtcagtct tcctcttacc cccaaaaccc aaggacaccc tcatgatctc ccggacccct  780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg  840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgccggagga gcagtacaac  900agcacgctcc gtgtggtcag catcctcacc gtcctgcacc aggactggct gaatggcaag  960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctctcgtg 1200ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc tccgggtaaa tga 1353Heavy chain having FcMT1 variant Fc region amino acid sequence(SEQ ID NO: 9):QVQLQQSGPE LVKPGASLKL SCTASGFNIK DTYIHWVKQR PEQGLEWIGRIYPTNGYTRY  60DPKFQDKATI TADTSSNTAY LQVSRLTSED TAVYYCSRWG GDGFYAMDYWGQGASVTVSS 120ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS 180GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCPPCPAPELLGG 240PSVFLLPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNAKTKPPEEQYN 300STLRVVSILT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSRDE 360LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPLV LDSDGSFFLYSKLTVDKSRW 420QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450Heavy chain having FcMT2 variant Fc region nucleic acid sequence(SEQ ID NO: 10):caggttcagc tgcagcagtc tggccctgag ctggtgaagc caggggcctc actcaagttg   60tcctgtacag cttctggctt caacatcaaa gacacctata tccactgggt gaaacagagg  120cctgaacagg gcctggaatg gattggaagg atttatccta ccaatggcta tactagatat  180gacccaaagt tccaggacaa ggccactatc acagcagaca catcctccaa cacagcctac  240ctgcaagtca gccgcctgac atctgaggac actgccgtct attactgctc ccggtgggga  300ggggacggct tctatgctat ggactactgg ggtcagggag cctccgtgac cgtgagctcc  360gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg  420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg  480tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca  540ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc  600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc  660aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cgtgggggga  720ccgtcagtct tcctcttacc cccaaaaccc aaggacaccc tcatgatctc ccggacccct  780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg  840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgccggagga gcagtacaac  900agcacgctcc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag  960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctctcgtg 1200ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc tccgggtaaa tga 1353Heavy chain having FcMT2 variant Fc region amino acid sequence(SEQ ID NO: 11):QVQLQQSGPE LVKPGASLKL SCTASGFNIK DTYIHWVKQR PEQGLEWIGR IYPTNGYTRY  60DPKFQDKATI TADTSSNTAY LQVSRLTSED TAVYYCSRWG GDGFYAMDYW GQGASVTVSS 120ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 180GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELVGG 240PSVFLLPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPPEEQYN 300STLRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 360LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPLV LDSDGSFFLY SKLTVDKSRW 420QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450Heavy chain having FcMT3 variant Fc region nucleic acid sequence(SEQ ID NO: 12):caggttcagc tgcagcagtc tggccctgag ctggtgaagc caggggcctc actcaagttg   60tcctgtacag cttctggctt caacatcaaa gacacctata tccactgggt gaaacagagg  120cctgaacagg gcctggaatg gattggaagg atttatccta ccaatggcta tactagatat  180gacccaaagt tccaggacaa ggccactatc acagcagaca catcctccaa cacagcctac  240ctgcaagtca gccgcctgac atctgaggac actgccgtct attactgctc ccggtgggga  300ggggacggct tctatgctat ggactactgg ggtcagggag cctccgtgac cgtgagctcc  360gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg  420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg  480tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca  540ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc  600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc  660aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga  720ccgtcagtct tcctcttacc cccaaaaccc aaggacaccc tcatgatctc ccggacccct  780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg  840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgccggagga gcagtacaac  900agcacgctcc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag  960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc tccgggtaaa tga 1353Heavy chain having FcMT3 variant Fc region amino acid sequence(SEQ ID NO: 13):QVQLQQSGPE LVKPGASLKL SCTASGFNIK DTYIHWVKQR PEQGLEWIGR IYPTNGYTRY  60DPKFQDKATI TADTSSNTAY LQVSRLTSED TAVYYCSRWG GDGFYAMDYW GQGASVTVSS 120ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 180GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG 240PSVFLLPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPPEEQYN 300STLRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 360LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450

In one embodiment, a variant 4D5 heavy chain has a modification in theFc region, and is encoded by the nucleic acid sequence of SEQ ID NO: 8or comprises the amino acid sequence of SEQ ID NO: 9, or is encoded bythe nucleic acid sequence of SEQ ID NO: 10 or comprises the amino acidsequence of SEQ ID NO: 11, or is encoded by the nucleic acid sequence ofSEQ ID NO: 12 or comprises the amino acid sequence of SEQ ID NO: 13.Preferably, the antibodies comprise a heavy chain encoded by a nucleicacid sequence selected from the group consisting of SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, and SEQ ID NO: 12, or comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, and SEQ ID NO: 13.

In one embodiment, an anti-HER2/neu antibody comprises an immunoglobulinlight chain having a N65S modification in the V_(L) region, and animmunoglobulin heavy chain having a modified Fc region. Preferably, ananti-HER2/neu antibody comprises a light chain having the amino acidsequence of SEQ ID NO: 2, and a heavy chain having an amino acidsequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, and SEQ ID NO: 13. In some embodiments, a polypeptideof the invention further comprises a light chain constant domain fusedto a light chain variable domain, which in some embodiments comprises atleast SEQ ID NO: 4. In other embodiments, the antibody is modified, afragment, or a modified fragment.

Chimeric 4D5 antibodies were constructed in accordance with the variousembodiments of the invention, to enhance binding to activatinglow-affinity Fc receptors, and to not alter, or only minimally increase,binding to the low-affinity inhibitor receptor CD32B (FcγRII-B). Theantibodies include the following wild-type and Fc-optimized antibodies:

-   ch4D5-wild-type Fc, which has a light chain having an amino acid    sequence of SEQ ID NO: 2, and a heavy chain having an amino acid    sequence of SEQ ID NO: 7. ch4D5-wild-type Fc has an N65S    substitution on the light chain, which results in a de-glycosylated    light chain.-   ch4D5-FcMT1, which has a light chain having an amino acid sequence    of SEQ ID NO: 2, and a heavy chain having an amino acid sequence of    SEQ ID NO: 9. ch4D5-FcMT1 has an N65S substitution on the light    chain, which results in a de-glycosylated light chain, and F243L,    R292P, Y300L, V305I, and P396L substitutions on the heavy chain (all    numbered according to Kabat). ch4D5-FcMT1 exhibits a 10-fold    increase in binding to human CD16A (FcγRIII-A), and binding to    CD16-158^(Phe) is enhanced in a proportionally greater fashion than    binding to CD16-158^(Val).-   ch4D5-FcMT2, which has a light chain having an amino acid sequence    of SEQ ID NO: 2, and a heavy chain having an amino acid sequence of    SEQ ID NO: 11. ch4D5-FcMT2 has an N65S substitution on the light    chain, which results in a de-glycosylated light chain, and L235V,    F243L, R292P, Y300L, and P396L substitutions on the heavy chain (all    numbered according to Kabat). This antibody is a further refinement    of the ch4D5-FcMT1 antibody, and has similar CD16A binding    properties, but also has a more favorable reduction in binding to    CD32B (FcγRII-B).-   ch4D5-FcMT3, which has a light chain having an amino acid sequence    of SEQ ID NO: 2, and a heavy chain having an amino acid sequence of    SEQ ID NO: 13. ch4D5-FcMT3 has an N65S substitution on the light    chain, which results in a de-glycosylated light chain, and F243L,    R292P, and Y300L substitutions on the heavy chain (all numbered    according to Kabat). This antibody is a further refinement of the    ch4D5-FcMT1 antibody, and has similar CD16A binding properties, but    also has a more favorable reduction in binding to CD32B (FcγRII-B).-   ch4D5-Ag-   ch4D5-N297Q, which has a light chain having an amino acid sequence    of SEQ ID NO: 2, and a heavy chain having an N297Q substitution    (numbered according to Kabat).

A comparison of the heavy chain sequences of the ch4D5-wild-type Fc andthe Fc-optimized variants ch4D5-FcMT1, ch4D5-FcMT2, and ch4D5-FcMT3 isshown in FIG. 2. The CDRs are indicated with black bars shown underneaththe pertinent residues.

Polypeptides (especially antibodies) contemplated by the presentinvention may comprise all or part of any amino acid sequence of thepresent invention. For example, in one embodiment a polypeptidecomprises a murine 4D5 immunoglobulin light chain having an N65Ssubstitution, and in another embodiment a polypeptide comprises a murine4D5 immunoglobulin heavy chain variant. In another embodiment, apolypeptide comprises an immunoglobulin light chain variable domainhaving the amino acid sequence of SEQ ID NO: 4, or an immunoglobulinlight chain having the amino acid sequence of SEQ ID NO: 2. In anotherembodiment, the polypeptide comprises a variant Fc domain, or animmunoglobulin heavy chain having the amino acid sequence of SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or a 4D5immunoglobulin heavy chain comprising F243L, R292P, and Y300Lsubstitutions.

Polypeptides (especially antibodies) contemplated by the presentinvention may be in a complex with one another or with othernon-immunoglobulin polypeptides (e.g., enzymes, hormones, structuralproteins, etc.). For example, an embodiment may provide a polypeptidecomplex comprising two polypeptides, wherein one of said polypeptidescomprises a heavy chain, and the other polypeptide comprises a variantlight chain, or wherein both polypeptides comprise the same sequences.Complexing can be mediated by any suitable technique, including bydimerization/multimerization at a dimerization/multimerization domainsuch as those described herein or covalent interactions (such as througha disulfide linkage) (which in some contexts is part of a dimerizationdomain, for example a dimerization domain may contain a leucine zippersequence and a cysteine). In another embodiment, a composition maycomprise polypeptides and/or polynucleotides of the invention, forexample a composition may comprise a plurality of any of thepolypeptides described herein. A composition comprising a polynucleotideor polypeptide may be in the form of a kit or an article of manufacture(optionally packaged with instructions, buffers, etc.).

It is also contemplated that polypeptide variants (and in particularantibody variants) can be prepared. The polypeptide variants may possesssequence modifications (e.g., substitutions, deletions and/or additions)at desired positions within their amino acid sequences relative to thenative amino acid sequence. Those skilled in the art will appreciatethat amino acid changes may alter post-translational processes of theantibody or polypeptide, such as changing the number or position ofglycosylation sites or altering the membrane anchoring characteristics.In a preferred embodiment, the antibody and polypeptide variants are Fcregion variants.

Variants may have the same or altered activity as compared to a nativeantibody or polypeptide. For example, it may be desirable that thevariant have the same activity, but be modified in a manner so that itis more stable or has a longer half-life in vivo, for example byconjugating the antibody with albumin or a salvage receptor bindingepitope, as described, e.g., in U.S. Pat. No. 5,739,277. Or, forexample, it may be desirable that an antibody have an increased bindingaffinity to antigen, but the same effector function as a nativeantibody, or it may be desirable that an antibody have the same bindingaffinity to antigen, but a decreased effector function. Activity may betested by, e.g., using in vitro assays such as ELISA assays, surfaceplasmon resonance assays, radiolabeled protein binding assays (RIA), orimmunoprecipitation assays.

Substantial modifications in function or immunological identity may beaccomplished by selecting modifications that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the modification, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Scanning aminoacid analysis can also be employed to identify one or more amino acidsalong a contiguous sequence, for example as described by Cunningham andWells (1989) Science 244:1081-1085. Among the preferred scanning aminoacids are relatively small, neutral amino acids, such as alanine,glycine, serine, and cysteine. Alanine is typically a preferred scanningamino acid among this group because it is the most common amino acid, isfrequently found in both buried and exposed positions, and because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. If alaninesubstitution does not yield adequate amounts of variant, an isotericamino acid can be used. Further, any cysteine residue not involved inmaintaining the proper conformation of the antibody or polypeptide maybe substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking However, incertain circumstances, particularly where the antibody is an antibodyfragment such as an Fv fragment, cysteine bond(s) may be added to theantibody or polypeptide to improve its stability.

B1. Fc Domain Variants

The polypeptides of the present invention may have variant Fc domains.Modification of the Fc domain normally leads to an altered phenotype,for example altered serum half-life, altered stability, alteredsusceptibility to cellular enzymes or altered effector function. It maybe desirable to modify the antibody of the invention with respect toeffector function, so as to enhance the effectiveness of the antibody intreating cancer, for example Reduction or elimination of effectorfunction is desirable in certain cases, for example in the case ofantibodies whose mechanism of action involves blocking or antagonism,but not killing of the cells bearing a target antigen. Increasedeffector function is generally desirable when directed to undesirablecells, such as tumor and foreign cells, where the FcγRs are expressed atlow levels, for example, tumor specific B cells with low levels ofFcγRIIB (e.g., non-Hodgkins lymphoma, CLL, and Burkitt's lymphoma). Insaid embodiments, molecules of the invention with conferred or alteredeffector function activity are useful for the treatment and/orprevention of a disease, disorder or infection where an enhancedefficacy of effector function activity is desired.

In certain embodiments, the molecules of the invention comprise one ormore modifications to the amino acids of the Fc domain, which reduce theaffinity and avidity of the Fc region and, thus, the molecule of theinvention, for one or more FcγR receptors. In other embodiments, themolecules of the invention comprise one or more modifications to theamino acids of the Fc region, which increase the affinity and avidity ofthe Fc region and, thus, the molecule of the invention, for one or moreFcγR receptors. In other embodiments, the molecules comprise a variantFc domain wherein said variant confers or mediates increased ADCCactivity and/or an increased binding to FcγRIIA, relative to a moleculecomprising no Fc domain or comprising a wild-type Fc domain. Inalternate embodiments, the molecules comprise a variant Fc domainwherein said variant confers or mediates decreased ADCC activity (orother effector function) and/or an increased binding to FcγRIIB,relative to a molecule comprising no Fc domain or comprising a wild-typeFc domain.

In some embodiments, the invention encompasses molecules comprising avariant Fc region, which variant Fc region does not show a detectablebinding to any FcγR, relative to a comparable molecule comprising thewild-type Fc region. In other embodiments, the invention encompassesmolecules comprising a variant Fc region, which variant Fc region onlybinds a single FcγR, preferably one of FcγRIIA, FcγRIIB, or FcγRIIIA.

The polypeptides of the present invention may comprise alteredaffinities for an activating and/or inhibitory Fcγ receptor. In oneembodiment, the antibody or polypeptide comprises a variant Fc regionthat has increased affinity for FcγRIIB and decreased affinity forFcγRIIIA and/or FcγRIIA, relative to a comparable molecule with awild-type Fc region. In another embodiment, the polypeptides of thepresent invention comprise a variant Fc region, which has decreasedaffinity for FcγRIIB and increased affinity for FcγRIIIA and/or FcγRIIA,relative to a comparable molecule with a wild-type Fc region. In yetanother embodiment, the polypeptides of the present invention comprise avariant Fc region that has decreased affinity for FcγRIIB and decreasedaffinity for FcγRIIIA and/or FcγRIIA, relative to a comparable moleculewith a wild-type Fc region. In still another embodiment, thepolypeptides of the present invention comprise a variant Fc region,which has unchanged affinity for FcγRIIB and decreased (or increased)affinity for FcγRIIIA and/or FcγRIIA, relative to a comparable moleculewith a wild-type Fc region.

In certain embodiments, the invention encompasses immunoglobulinscomprising a variant Fc region with an altered affinity for FcγRIIIAand/or FcγRIIA such that the immunoglobulin has an enhanced effectorfunction, e.g., antibody dependent cell mediated cytotoxicity.Non-limiting examples of effector cell functions includeantibody-dependent cell mediated cytotoxicity (ADCC), antibody-dependentphagocytosis, phagocytosis, opsonization, opsonophagocytosis, cellbinding, rosetting, C1q binding, and complement dependent cell mediatedcytotoxicity.

In a preferred embodiment, the alteration in affinity or effectorfunction is at least 2-fold, preferably at least 4-fold, at least5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least9-fold, at least 10-fold, at least 50-fold, or at least 100-fold,relative to a comparable molecule comprising a wild-type Fc region. Inother embodiments of the invention, the variant Fc regionimmunospecifically binds one or more FcRs with at least 65%, preferablyat least 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%,225%, or 250% greater affinity relative to a molecule comprising awild-type Fc region. Such measurements can be in vivo or in vitroassays, and in a preferred embodiment are in vitro assays such as ELISAor surface plasmon resonance assays.

In different embodiments, the molecules comprise a variant Fc domainwherein said variant agonizes at least one activity of an FcγR receptor,or antagonizes at least one activity of an FcγR receptor. In a preferredembodiment, the molecules comprise a variant that agonizes (orantagonizes) one or more activities of FcγRIIB, for example, B cellreceptor-mediated signaling, activation of B cells, B cellproliferation, antibody production, intracellular calcium influx of Bcells, cell cycle progression, FcγRIIB-mediated inhibition of FcεRIsignaling, phosphorylation of FcγRIIB, SHIP recruitment, SHIPphosphorylation and association with Shc, or activity of one or moredownstream molecules (e.g., MAP kinase, JNK, p38, or Akt) in the FcγRIIBsignal transduction pathway. In another embodiment, the moleculescomprise a variant that agonizes (or antagonizes) one or more activitiesof FcεRI, for example, mast cell activation, calcium mobilization,degranulation, cytokine production, or serotonin release.

In certain embodiments, the molecules comprise an Fc domain comprisingdomains or regions from two or more IgG isotypes (e.g., IgG1, IgG2, IgG3and IgG4). The various IgG isotypes exhibit differing physical andfunctional properties including serum half-life, complement fixation,FcγR binding affinities and effector function activities (e.g. ADCC,CDC, etc.) due to differences in the amino acid sequences of their hingeand/or Fc domains, for example as described in Flesch and Neppert (1999)J. Clin. Lab. Anal. 14:141-156; Chappel et al. (1993) J. Biol. Chem.33:25124-25131; Chappel et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.)88:9036-9040; Brüggemann et al. (1987) J. Exp. Med 166:1351-1361. Thistype of variant Fc domain may be used alone, or in combination with anamino acid modification, to affect Fc-mediated effector function and/orbinding activity. In combination, the amino acid modification and IgGhinge/Fc region may display similar functionality (e.g., increasedaffinity for FcγRIIA) and may act additively or, more preferably,synergistically to modify the effector functionality in the molecule ofthe invention, relative to a molecule of the invention comprising awild-type Fc region. In other embodiments, the amino acid modificationand IgG Fc region may display opposite functionality (e.g., increasedand decreased affinity for FcγRIIA, respectively) and may act toselectively temper or reduce a specific functionality in the molecule ofthe invention, relative to a molecule of the invention not comprising anFc region or comprising a wild-type Fc region of the same isotype.

In a preferred specific embodiment, the molecules comprise a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculehas an altered affinity for an FcR, provided that said variant Fc regiondoes not have a substitution at positions that make a direct contactwith FcγR based on crystallographic and structural analysis of Fc-FcRinteractions such as those disclosed by Sondermann et al. (2000) Nature406:267-273. Examples of positions within the Fc region that make adirect contact with FcγR are amino acid residues 234-239 (hinge region),amino acid residues 265-269 (B/C loop), amino acid residues 297-299(C′/E loop), and amino acid residues 327-332 (F/G loop). In someembodiments, the molecules of the invention comprise variant Fc regionscomprise modification of at least one residue that does not make adirect contact with an FcγR based on structural and crystallographicanalysis, e.g., is not within the Fc-FcγR binding site.

Variant Fc domains are well known in the art, and any known Fc variantmay be used in the present invention to confer or modify the effectorfunction exhibited by a molecule of the invention comprising an Fcdomain (or portion thereof) as functionally assayed, e.g., in an NKdependent or macrophage dependent assay. For example, Fc domain variantsidentified as altering effector function are disclosed in the AntibodyEngineering Technology Art, and any suitable variant disclosed thereinmay be used in the present molecules.

In certain embodiments, the molecules comprise a variant Fc region,having one or more amino acid modifications in one or more regions,which modification(s) alter (relative to a wild-type Fc region) theRatio of Affinities of the variant Fc region to an activating FcγR (suchas FcγRIIA or FcγRIIIA) relative to an inhibiting FcγR (such asFcγRIIB):

${{Ratio}\mspace{14mu} {of}\mspace{14mu} {Affinities}} = \frac{{Wild}\text{-}{Type}\mspace{14mu} {to}\mspace{14mu} {Variant}\mspace{14mu} {Change}\mspace{14mu} {in}\mspace{14mu} {Affinity}\mspace{14mu} {to}\mspace{14mu} {Fc}\; \gamma \; R_{Activating}}{{Wild}\text{-}{Type}\mspace{14mu} {to}\mspace{14mu} {Variant}\mspace{14mu} {Change}\mspace{14mu} {in}\mspace{14mu} {Affinity}\mspace{14mu} {to}\mspace{14mu} {Fc}\; \gamma \; R_{Inhibiting}}$

Where an Fc variant has a Ratio of Affinities greater than 1, themethods of the invention have particular use in providing a therapeuticor prophylactic treatment of a disease, disorder, or infection, or theamelioration of a symptom thereof, where an enhanced efficacy ofeffector cell function (e.g., ADCC) mediated by FcγR is desired, e.g.,cancer or infectious disease. Where an Fc variant has a Ratio ofAffinities less than 1, the methods of the invention have particular usein providing a therapeutic or prophylactic treatment of a disease ordisorder, or the amelioration of a symptom thereof, where a decreasedefficacy of effector cell function mediated by FcγR is desired, e.g.,autoimmune or inflammatory disorders. Table 2 lists exemplary single,double, triple, quadruple and quintuple mutations by whether their Ratioof Affinities is greater than or less than 1. Specific binding data forvarious mutations is listed in Table 3, and more information concerningthese mutations may be found in the Antibody Engineering Technology Art.

TABLE 2 Exemplary Single and Multiple Mutations Listed by Ratio ofAffinities Ratio Single Double Triple Quadruple Quintuple >1 F243L F243L& F243L, P247L & L234F, F243L, R292P & L235V, F243L, D270E R292P N421KY300L R292P, Y300L R292G F243L & F243L, R292P & L235I, F243L, R292P & &P396L R292P Y300L Y300L Y300L L235P, F243L, F243L & F243L, R292P &L235Q, F243L, R292P & R292P, Y300L P396L V305I Y300L & P396L D270E &F243L, R292P & F243L, P247L, D270E & F243L, R292P, P396L P396L N421KV305I, Y300L R292P & F243L, Y300L & F243L, R255L, D270E & & P396L Y300LP396L P396L R292P & P247L, D270E & F243L, D270E, G316D V305I N421K &R416G R292P & R255L, D270E & F243L, D270E, K392T P396L P396L & P396LY300L & D270E, G316D & F243L, D270E, P396L & P396L R416G Q419H P396L &D270E, K392T & F243L, R292P, Y300L, Q419H P396L & P396L D270E, P396L &F243L, R292P, V305I & Q419H P396L V284M, R292L & P247L, D270E, Y300LK370N & N421K R292P, Y300L & R255L, D270E, R292G P396L & P396L R255L,D270E, Y300L & P396L D270E, G316D, P396L & R416G <1 Y300L F243L & F243L,R292P & P396L P396L V305I P247L & N421K R255L & P396L R292P & V305IK392T & P396L P396L & Q419H

TABLE 3 Detailed Binding Information for Exemplary Fc Variants Ratio ofAffinities CD16A CD16A CD16A/CD32B Fc sequence V158 F158 CD32B V158 F158Ratio of Affinities >1 Class I: Increased Binding to CD16; DecreasedBinding to CD32B F243L 4.79 3.44 0.84 5.70 4.10 F243L P247L D270E 2.303.45 0.32 7.19 10.78 N421K F243L P247L N421K 1.89 1.71 0.17 11.12 10.06F243L R255L D270E 1.75 1.64 0.38 4.61 4.32 P396L F243L D270E G316D 1.501.34 0.20 7.50 6.70 R416G F243L D270E K392T 3.16 2.44 0.44 7.18 5.55P396L F243L D270E P396L 1.46 1.15 0.26 5.62 4.42 Q419H F243L R292P 4.730.12 39.4 F243L R292P 4 1.67 0.16 25 10.44 F243L R292P P300L 6.69 2.30.32 20.9 7.19 F243L R292P V305I 2.56 1.43 ND >25 >25 F243L R292P V305I5.37 2.53 0.40 13.43 6.33 P396L P247L D270E N421K 1.89 2.46 0.58 3.264.24 R255L D270E R292G 1.39 1.30 0.65 2.14 2.00 P396L R255L D270E Y300L1.52 1.74 0.87 1.75 2.00 P396L R255L D270E P396L 1.34 1.65 0.87 1.541.90 D270E 1.25 1.48 0.39 3.21 3.79 D270E G316D R416G 2.18 2.49 0.782.79 3.19 D270E K392T P396L 1.81 2.28 0.79 2.29 2.89 D270E P396L 1.381.65 0.89 1.55 1.85 D270E P396L G316D 1.22 1.07 1.14 R416G D270E P396LQ419H 1.64 2.00 0.68 2.41 2.94 V284M R292P K370N 1.14 1.37 0.37 3.1 3.7R292G 1.54 0.25 6.2 R292P 2.90 0.25 11.60 R292P V305I 1.32 1.28 0.37 3.63.46 Class II: Decreased Binding to CD16; Greatly Decreased Binding toCD32B R292P 0.64 0.25 2.56 R292P F243L 0.6 0.12 5.00 Class III:Increased Binding to CD16; Unchanged Binding to CD32B F243I R292P Y300L10.9 3.12 1.05 10.4 2.97 V305I P396L F243L R292P Y300L 10.06 5.62 1.079.40 5.25 P396L R292P V305I P396L 1.85 1.90 0.92 2.01 2.07 Class IV:Greatly Increased Binding to CD16; Increased Binding to CD32B F243LR292P Y300L 10.06 8.25 1.38 7.29 5.98 V305I P396L D270E G316D P396L 1.221.07 1.14 R416G Ratio of Affinities <1 Class V: Unchanged Binding toCD16; Increased Binding to CD32B R255L P396L 1.09 2.22 0.49 Y300L 1.011.18 0.99 Class VI: Increased Binding to CD16; Greatly Increased Bindingto CD32B F243L P396L 1.49 1.60 2.22 0.67 0.72 P247L N421K 1.29 1.73 2.000.65 0.87 R255L P396L 1.39 2.22 0.49 0.63 R292P V305I 1.59 2.11 2.670.60 0.79 K392T P396L 1.49 1.81 2.35 0.63 0.77 P396L 1.27 1.73 2.58 0.490.67 P396L Q419H 1.19 1.19 1.33 0.89 0.89 Class VII: Decreased Bindingto CD16; Increased/Unchanged Binding to CD32B D270E G316D P396L 0.941.07 0.88 R416G

In other embodiments, the molecules comprise a variant Fc region havingone or more amino acid substitutions, which substitutions alter(relative to a wild-type Fc region) the binding of the variant Fcregion, e.g., enhance the binding to an activating FcγR (such as FcγRIIAor FcγRIIIA) and/or reduce the binding to an inhibiting FcγR (such asFcγRIIB). Various Fc mutations having one or more amino acid changeswere engineered and analyzed by surface plasmon resonance for k_(off),as shown in Table 4. Dissociation rate constants for binding the variousFcγR were determined by BIAcore analysis and directly compared withthose for the wild-type Fc, with the ratio (x=WT k_(off)/mutant k_(off))indicated in the right-hand columns of Table 4 with respect to each FcγRtested.

There is also extensive guidance in the Antibody Engineering TechnologyArt concerning desirable modifications. Exemplary modifications that maybe desirable in certain circumstances are listed below:

In a specific embodiment, in variant Fc regions, any amino acidmodifications (e.g., substitutions) at any of positions 235, 240, 241,243, 244, 247, 262, 263, 269, 298, 328, or 330 and preferably one ormore of the following residues: A240, I240, L241, L243, H244, N298, I328or V330. In a different specific embodiment, in variant Fc regions, anyamino acid modifications (e.g., substitutions) at any of positions 268,269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305,307, 309, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419,430, 434, 435, 437, 438 or 439 and preferably one or more of thefollowing residues: H280, Q280, Y280, G290, S290, T290, Y290, N294,K295, P296, D298, N298, P298, V298, I300 or L300.

In a preferred embodiment, in variant Fc regions that bind an FcγR withan altered affinity, any amino acid modifications (e.g., substitutions)at any of positions 255, 256, 258, 267, 268, 269, 270, 272, 276, 278,280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301,303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331, 333, 334,335, 337, 338, 339, 340, 359, 360, 373, 376, 416, 419, 430, 434, 435,437, 438 or 439. Preferably, the variant Fc region has any of thefollowing residues: A256, N268, Q272, D286, Q286, S286, A290, S290,A298, M301, A312, E320, M320, Q320, R320, E322, A326, D326, E326, N326,S326, K330, T339, A333, A334, E334, H334, L334, M334, Q334, V334, K335,Q335, A359, A360 or A430.

In a different embodiment, in variant Fc regions that bind an FcγR (viaits Fc region) with a reduced affinity, any amino acid modifications(e.g., substitutions) at any of positions 252, 254, 265, 268, 269, 270,278, 289, 292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324, 327,329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434,435, 437, 438 or 439.

In a different embodiment, in variant Fc regions that bind an FcγR (viaits Fc region) with an enhanced affinity, any amino acid modifications(e.g., substitutions) at any of positions 280, 283, 285, 286, 290, 294,295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340,360, 378, 398 or 430. In a different embodiment, in variant Fc regionsthat binds FcγRIIA with an enhanced affinity, any of the followingresidues: A255, A256, A258, A267, A268, N268, A272, Q272, A276, A280,A283, A285, A286, D286, Q286, S286, A290, S290, M301, E320, M320, Q320,R320, E322, A326, D326, E326, S326, K330, A331, Q335, A337 or A430.

In other embodiments, the invention encompasses the use of any Fcvariant known in the art, such as those disclosed in Jefferis et al.(2002) Immunol Lett 82:57-65; Presta et al. (2002) Biochem Soc Trans30:487-90; Idusogie et al. (2001) J Immunol 166:2571-75; Shields et al.(2001) J Biol Chem 276:6591-6604; Idusogie et al. (2000) J Immunol164:4178-84; Reddy et al. (2000) J Immunol 164:1925-33; Xu et al. (2000)Cell Immunol 200:16-26; Armour et al. (1999) Eur J Immunol 29:2613-24;Jefferis et al. (1996) Immunol Lett 54:101-04; Lund et al. (1996) JImmunol 157:4963-69; Hutchins et al. (1995) Proc. Natl. Acad. Sci.(U.S.A.) 92:11980-84; Jefferis et al. (1995) Immunol Lett. 44:111-17;Lund et al. (1995) FASEB J 9:115-19; Alegre et al. (1994)Transplantation 57:1537-43; Lund et al. (1992) Mol Immunol 29:53-59;Lund et al. (1991) J. Immunol 147:2657-62; Duncan et al. (1988) Nature332:563-64; U.S. Pat. Nos. 5,624,821; 5,885,573; 6,194,551; 7,276,586;and 7,317,091; and PCT Publications WO 00/42072 and PCT WO 99/58572.

Preferred variants include one or more modifications at any ofpositions: 228, 230, 231, 232, 233, 234, 235, 239, 240, 241, 243, 244,245, 247, 262, 263, 264, 265, 266, 271, 273, 275, 281, 284, 291, 296,297, 298, 299, 302, 304, 305, 313, 323, 325, 326, 328, 330 or 332.

Particularly preferred variants include one or more modificationsselected from groups A-AI:

-   A. 228E, 228K, 228Y or 228G;-   B. 230A, 230E, 230Y or 230G;-   C. 231E, 231K, 231Y, 231P or 231G;-   D. 232E, 232K, 232Y, 232G;-   E. 233D;-   F. 2341 or 234F;-   G. 235D, 235Q, 235P, 235I or 235V;-   H. 239D, 239E, 239N or 239Q;-   I. 240A, 240I, 240M or 240T;-   J. 243R, 243, 243Y, 243L, 243Q, 243W, 243H or 243I;-   K. 244H;-   L. 245A;-   M. 247G, 247V or 247L;-   N. 262A, 262E, 262I, 262T, 262E or 262F;-   O. 263A, 263I, 263M or 263T;-   P. 264F, 264E, 264R, 264I, 264A, 264T or 264W;-   Q. 265F, 265Y, 265H, 265I, 265L, 265T, 265V, 265N or 265Q;-   R. 266A, 266I, 266M or 266T;-   S. 271D, 271E, 271N, 271Q, 271K, 271R, 271S, 271T, 271H, 271A, 271V,    271L, 271I, 271F, 271M, 271Y, 271W or 271G;-   T. 273I;-   U. 275L or 275W;-   V. 281D, 281K, 281Y or 281P;-   W. 284E, 284N, 284T, 284L, 284Y or284M;-   X. 291D, 291E, 291Q, 291T, 291H, 291I or 291G;-   Y. 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M, 299N,    299P, 299Q, 299R, 299S, 299V, 299W or 299Y;-   Z. 302I;-   AA. 304D, 304N, 304T, 304H or 304L-   AB. 305I;-   AC. 313F;-   AD. 323I;-   AE. 325A, 325D, 325E, 325G, 325H, 325I, 325L, 325K, 325R, 325S,    325F, 325M, 325T, 325V, 325Y, 325W or 325P;-   AF. 328D, 328Q, 328K, 328R, 328S, 328T, 328V, 328I, 328Y, 328W,    328P, 328G, 328A, 328E, 328F, 328H, 328M or 328N;-   AG. 330L, 330Y, 330I or 330V;-   AH. 332A, 332D, 332E, 332H, 332N, 332Q, 332T, 332K, 332R, 332S,    332V, 332L, 332F, 332M, 332W, 332P, 332G or 332Y; and-   AI. 336E, 336K or 336Y.

Still more particularly preferred variants include one or moremodifications selected from Groups 1-105:

Group Variant 1 A330L/I332E 2 D265F/N297E/I332E 3 D265Y/N297D/I332E 4D265Y/N297D/T299L/I332E 5 F241E/F243Q/V262T/V264F 6F241E/F243Q/V262T/V264E/I332E 7 F241E/F243R/V262E/V264R 8F241E/F243R/V262E/V264R/I332E 9 F241E/F243Y/V262T/V264R 10F241E/F243Y/V262T/V264R/ I332E 11 F241L/F243L/V262I/V264I 12 F241L/V262I13 F241R/F243Q/V262T/V264R 14 F241R/F243Q/V262T/V264R/ I332E 15F241W/F243W/V262A/V264A 16 F241Y/F243Y/V262T/V264T 17F241Y/F243Y/V262T/V264T/ N297D/I332E 18 F243L/V262I/V264W 19 P243L/V264I20 L328D/I332E 21 L328E/I332E 22 L328H/I332E 23 L328I/I332E 24L328M/I332E 25 L328N/I332E 26 L328Q/I332E 27 L328T/I332E 28 L328V/I332E29 N297D/A330Y/I332E 30 N297D/I332E 31 N297D/I332E/S239D/A330L 32N297D/S298A/A330Y/I332E 33 N297D/T299L/I332E 34 N297D/T299F/I332E/N297D/T299H/I332E 35 N297D/T299I/I332E 36 N297D/T299L/I332E 37N297D/T299V/I332E 38 N297E/I332E 39 N297S/I332E 40 P230A/E233D/I332E 41P244H/P245A/P247V 42 S239D/A330L/I332E 43 S239D/A330Y/I332E 44S239D/A330Y/I332E/K326E 45 S239D/A330Y/I332E/K326T 46S239D/A330Y/I332E/L234I 47 S239D/A330Y/I332E/L235D 48S239D/A330Y/I332E/V240I 49 S239D/A330Y/I332E/V264T 50S239D/A330Y/I332E/V266I 51 S239D/D265F/N297D/I332E 52S239D/D265H/N297D/I332E 53 S239D/D265I/N297D/I332E 54S239D/D265L/N297D/I332E 55 S239D/D265T/N297D/I332E 56S239D/D265V/N297D/I332E 57 S239D/D265Y/N297D/I332E 58 S239D/I332D 59S239D/I332E 60 S239D/I332E/A330I 61 S239D/I332N 62 S239D/I332Q 63S239D/N297D/I332E 64 S239D/N297D/I332E/A330Y 65S239D/N297D/I332E/A330Y/F241S/ F243H/V262T/V264T 66S239D/N297D/I332E/K326E 67 S239D/N297D/I332E/L235D 68 S239D/S298A/I332E69 S239D/V264I/A330L/I332E 70 S239D/V264I/I332E 71S239D/V264I/S298A/I332E 72 S239E/D265N 73 S239E/D265Q 74 S239E/I332D 75S239E/I332E 76 S239E/I332N 77 S239E/I332Q 78 S239E/N297D/I332E 79S239E/V264I/A330Y/I332E 80 S239E/V264I/I332E 81S239E/V264I/S298A/A330Y/I332E 82 S239N/A330L/I332E 83 S239N/A330Y/I332E84 S239N/I332D 85 S239N/I332E 86 S239N/I332N 87 S239N/I332Q 88S239N1S298A/I332E 89 S239Q/I332D 90 S239Q/I332E 91 S239Q/I332N 92S239Q/I332Q 93 S239Q/V264I/I332E 94 S298A/I332E 95 V264E/N297D/I332E 96V264I/A330L/I332E 97 V264I/A330Y/I332E 98 V264I/I332E 99V264I/S298A/I332E 100 Y296D/N297D/I332E 101 Y296E/N297D/I332E 102Y296H/N297D/I332E 103 Y296N/N297D/I332E 104 Y296Q/N297I/I332E 105Y296T/N297D/I332E.

Effector function can be modified by techniques such as those describedin the Antibody Engineering Technology Art, or by other means. Forexample, cysteine residue(s) may be introduced in the Fc region, therebyallowing interchain disulfide bond formation in this region, resultingin the generation of a homodimeric antibody that may have improvedinternalization capability and/or increased complement-mediated cellkilling and ADCC. See Caron et al. (1992) J. Exp Med. 176:1191-1195; andB. Shopes (1992) J. Immunol. 148:2918-2922. Homodimeric antibodies withenhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. (1993)Cancer Research 53:2560-2565. Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. Stevenson et al. (1989)Anti-Cancer Drug Design 3:219-230.

B2. Sequence Modifications

Generally, sequence modifications may be the substitution, deletion, oraddition of one or more residues in the antibody or polypeptide thatresults in a change in the amino acid sequence as compared to the nativesequence. Guidance in determining which amino acid residue may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence of the antibody orpolypeptide with that of homologous known protein molecules andminimizing the number of amino acid sequence changes made in regions ofhigh homology. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

Amino acid substitutions may involve the conservative ornon-conservative substitution of one or more residues. Suchsubstitutions are well-known in the art, for example a conservativesubstitution entails replacing an amino acid with another amino acidhaving similar structural and/or chemical properties, such as thereplacement of a leucine with a serine. Non-conservative substitutionsgenerally entail replacing an amino acid with another amino acid havingdifferent structural and/or chemical properties, for example an acidicamino acid (e.g., Glu or Asp) may be replaced with a basic amino acid(e.g., Lys or Asn).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., a humanized or human antibody), in order to obtain avariant antibody having improved biological properties relative to theparent antibody. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites are mutated to generate all possibleamino substitutions at each site, the antibody variants thus generatedare displayed on phage, and the phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity). Inorder to identify candidate hypervariable region sites for modification,alanine scanning mutagenesis can be performed to identify hypervariableregion residues contributing significantly to antigen binding.Alternatively, or additionally, it may be beneficial to analyze acrystal structure of the antigen-antibody complex to identify contactpoints between the antibody and its antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

The modification may also involve the incorporation (e.g., bysubstitution or addition) of unnatural amino acids, for example bymethods such as those described in, e.g., Wang et al. (2002) Chem. Comm.1:1-11; Wang et al. (2001) Science 292:498-500; and van Hest et al.(2001) Chem. Comm. 19:1897-1904. Alternative strategies focus on theenzymes responsible for the biosynthesis of amino acyl-tRNA, asdescribed in, e.g., Tang et al. (2001) J. Am. Chem. 123(44):11089-11090;and Kiick et al. (2001) FEBS Lett. 505(3):465.

In a preferred embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues havebeen modified. Additionally or alternatively, such modifications may becharacterized as having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 modified amino acidresidues. In a particularly preferred embodiment, at least 1 but no morethan 10 residues have been modified. Additionally or alternatively, suchmodifications may be characterized as having no more than 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 modified amino acid residues. Themodifications may be all substitutions, all deletions, all additions, orany combination of substitutions, deletions, or additions.

Nucleic acid molecules encoding amino acid sequence variants may beprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring amino acid sequence variants) or preparationby oligonucleotide-mediated (or site-directed) mutagenesis, restrictionselection mutagenesis, PCR mutagenesis, and cassette mutagenesis of anearlier prepared variant or a non-variant version of the antibody.

B3. Other Modifications

The polypeptide variants (especially antibody variants) of the presentinvention include analogs and derivatives that are modified, e.g., bythe covalent attachment of any type of molecule as long as such covalentattachment permits the antibody to retain its epitope bindingimmunospecificity. For example, but not by way of limitation, thederivatives and analogs of the antibodies include those that have beenfurther modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular antibody unit orother protein, etc. Any of numerous chemical modifications can becarried out by known techniques, including, but not limited to specificchemical cleavage, acetylation, formylation, metabolic synthesis in thepresence of tunicamycin, etc. Additionally, the analog or derivative cancontain one or more unnatural amino acids.

The antibodies and polypeptides may be modified by introducing one ormore glycosylation sites into the antibodies, deleting one or moreglycosylation sites from the antibodies, or shifting an existingglycosylation site on the antibodies, preferably without altering thedesired functionality of the antibodies, e.g., binding activity.Glycosylation sites may be introduced into, or deleted from, thevariable and/or constant region of the antibodies, by methods known inthe art. For example, a glycosylation site may be introduced into anantibody of the invention by modifying or mutating an amino acidsequence of the antibody so that the desired sequence (e.g.,Asn-X-Thr/Ser) is obtained, and a glycosylation site may be shifted bymodifying position 296 in the Fc region, so that position 296 and notposition 297 is glycosylated. Methods of modifying the carbohydratecontent (glycosylation) of proteins are well known in the art, forexample as described in U.S. Pat. Nos. 6,472,511 and 6,218,149; U.S.Patent Publication Nos. 20030115614 and 20020028486; EP 0359096 B1; andWO 03/035835.

In some embodiments, molecules of the invention are engineered tocomprise an altered glycosylation pattern or an altered glycoform.Engineered glycoforms may be useful for a variety of purposes,including, but not limited to, enhancing effector function. Engineeredglycoforms may be generated by any method known to one skilled in theart, for example by using engineered or variant expression strains, byco-expression with one or more enzymes, for example,N-acetylglucosaminyltransferase III (GnT-III), by expressing an antibodyof the invention in various organisms or cell lines from variousorganisms, or by modifying carbohydrate(s) after the antibody has beenexpressed and purified. Methods for generating engineered glycoforms areknown in the art, and include but are not limited to those described in,e.g., Okazaki et al. (2004) JMB 336:1239-1249; Shinkawa et al. (2003) JBiol Chem 278:3466-3473; Shields et al. (2002) J Biol Chem277:26733-26740; Davies et al. (2001) Biotechnol Bioeng 74:288-294;Umana et al. (1999) Nat. Biotechnol 17:176-180; U.S. Pat. No. 6,602,684;U.S. Patent Publication Nos. 20030157108, 20030115614, and 20030003097;WO 02/311140; WO 02/30954; WO 01/292246; WO 00/61739; Potillegent™technology available from Biowa, Inc. (Princeton, N.J.); and GlycoMAb™glycosylation engineering technology available from GLYCARTbiotechnology AG (Zurich, Switzerland).

B4. Polypeptide Conjugates

The polypeptides of the present invention may be recombinantly fused orchemically conjugated (including both covalent and non-covalentconjugations) to heterologous polypeptides or portions thereof togenerate fusion proteins. Preferably, the polypeptide of the presentinvention (especially an antibody) is fused to at least 10, at least 15,at least 20, at least 25, at least 30, at least 40, at least 50, atleast 60, at least 70, at least 80, at least 90 or at least 100 aminoacids of the heterologous polypeptide to generate a desired fusionprotein. The fusion does not necessarily need to be direct, but mayoccur through linker sequences. Polypeptides of the present inventionmay also be attached to solid supports or semi-solid matrices, which areparticularly useful for immunoassays or purification of the targetantigen. Such supports and matrices include, but are not limited to,glass, cellulose, polyacrylamide, agarose beads, acrylamide beads,nylon, polystyrene, polyvinyl chloride or polypropylene. Attachment maybe accomplished, for example, by methods described in Methods inEnzymology, 44 (1976).

The antibodies and polypeptides may be conjugated to a therapeutic agentin order to modify a given biological response, affect (e.g., increase)the serum half-life of the therapeutic agent, or target the therapeuticagent to a particular subset of cells. They may also be fused to markersequences (e.g., a hexa-histidine peptide or a “flag” tag) to facilitatepurification. Techniques for conjugating such therapeutic moieties toantibodies are well known; see, e.g., Hellstrom et al., “Antibodies ForDrug Delivery”, in Controlled Drug Delivery (2nd ed., Robinson et al.(eds.), 1987, pp. 623-53, Marcel Dekker, Inc.).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of molecules of the invention (e.g.,antibodies with higher affinities and lower dissociation rates).Antibodies and polypeptides of the invention, or their encoding nucleicacids, may be further altered by being subjected to random mutagenesisby error-prone PCR, random nucleotide insertion or other methods priorto recombination. One or more portions of a polynucleotide encoding amolecule of the invention, may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

B5. Fragments

The invention additionally provides antibody and other polypeptidefragments. Such fragments may be truncated at the N-terminus orC-terminus, or may lack internal residues, for example, when comparedwith a full length native antibody or protein. Certain fragments maylack amino acid residues that are not essential for a desired biologicalactivity. These fragments may be prepared by any of a number ofconventional techniques. Desired peptide fragments may be chemicallysynthesized. An alternative approach involves generating antibody orpolypeptide fragments by enzymatic digestion, e.g., by treating theprotein with an enzyme known to cleave proteins at sites defined byparticular amino acid residues, or by digesting the DNA with suitablerestriction enzymes and isolating the desired fragment. Yet anothersuitable technique involves isolating and amplifying a DNA fragmentencoding a desired antibody or polypeptide fragment, by polymerase chainreaction (PCR). Oligonucleotides that define the desired termini of theDNA fragment are employed at the 5′ and 3′ primers in the PCR.Preferably, antibody and polypeptide fragments share at least onebiological and/or immunological activity with the native antibody orpolypeptide disclosed herein.

In some embodiments, a polypeptide of the invention further comprises adimerization domain, which can comprise a dimerization sequence, and/orsequence comprising one or more cysteine residues. In some embodiments,the dimerization domain will be located between an antibody heavy chainor light chain variable domain and at least a portion of a viral coatprotein, and one or more disulfide bond and/or a single dimerizationsequence may be present in the dimerization domain to provide forbivalent display. In some embodiments, heavy chains of a F(ab)₂ willdimerize at a dimerization domain not including a hinge region. Thedimerization domain may comprise a leucine zipper sequence.

In another embodiment, the polypeptide fragments of the presentinvention comprise an amino acid sequence of at least 5 contiguous aminoacid residues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 30contiguous amino acid residues, at least 40 contiguous amino acidresidues, at least 50 contiguous amino acid residues, at least 60contiguous amino residues, at least 70 contiguous amino acid residues,at least contiguous 80 amino acid residues, at least contiguous 90 aminoacid residues, at least contiguous 100 amino acid residues, at leastcontiguous 125 amino acid residues, at least 150 contiguous amino acidresidues, at least contiguous 175 amino acid residues, at leastcontiguous 200 amino acid residues, or at least contiguous 250 aminoacid residues of the amino acid sequence of another polypeptide. In aspecific embodiment, a fragment of a polypeptide retains at least onefunction of the polypeptide.

B6. Diabodies and DARTs

Diabodies and dual affinity retargeting reagents (“DARTs”) are alsoprovided by the present invention. The diabodies and DARTs compriseantigen binding domains generally derived from the antibodies andpolypeptides of the invention. The design and construction of diabodiesand DARTs is described in, for example, U.S. Provisional PatentApplication Nos. 61/019,051 filed on Jan. 4, 2008 and 60/945,523 filedon Jun. 21, 2007; U.S. patent application Ser. No. 11/409,339 filed onApr. 17, 2006; Marvin et al. (2005) Acta Pharmacol. Sin. 26:649-658;Olafsen et al. (2004) Prot. Engr. Des. Sel. 17:21-27; Holliger et al.(1993) Proc. Natl. Acad. Sci. (U.S.A.) 90:6444-6448. Each polypeptidechain of a diabody molecule comprises a V_(L) domain and a V_(H) domain,from the same or different antibodies, which are covalently linked suchthat the domains are constrained from self assembly. Interaction of twoof the polypeptide chains will produce two V_(L)-V_(H) pairings, formingtwo epitope binding sites, i.e., a bivalent molecule. Neither the V_(H)or V_(L) domain is constrained to any position within the polypeptidechain, nor are the domains restricted in their relative positions to oneanother; the only restriction is that a complementary polypeptide chainbe available in order to form functional diabody. The domains may beseparated by a peptide linker, and the polypeptide chains may beengineered to comprise at least one cysteine residue on each chain, sothat interchain disulfide bonds may be formed to stabilize the diabody.

Where the V_(L) and V_(H) domains are derived from the same antibody,the two complementary polypeptide chains may be identical, resulting ina bivalent monospecific antibody, or may be different, resulting in abivalent bispecific antibody (e.g., one that binds ton two differentepitopes on the same antigen). Where the V_(L) and V_(H) domains arederived from antibodies specific for different antigens, formation of afunctional bispecific diabody requires the interaction of two differentpolypeptide chains, i.e., formation of a heterodimer. In a particularembodiment, at least one epitope binding site of the diabody is specificfor an antigen on a particular cell, such as a B-cell or T-cell, aphagocytotic cell, a natural killer (NK) cell or a dendritic cell.

In various embodiments, one or more of the polypeptide chains of thediabody comprises an Fc domain. Fc domains in the polypeptide chains ofthe diabody molecules preferentially dimerize, resulting in theformation of a diabody molecule that exhibits immunoglobulin-likeproperties, e.g., Fc-FcγR interactions. Fc comprising diabodies may bedimers, e.g., comprised of two polypeptide chains, each comprising aV_(H) domain, a V_(L) domain and an Fc domain. In various embodiments,one or more of the polypeptide chains of the diabody comprises a hingedomain, which may be derived from any immunoglobulin isotype or allotypeincluding IgA, IgD, IgG, IgE and IgM. In preferred embodiments, thehinge domain is derived from IgG, wherein the IgG isotype is IgG1, IgG2,IgG3 or IgG4, or an allotype thereof. The hinge domain may be engineeredinto a polypeptide chain in any position relative to other domains orportions of the chain, and in certain circumstances may be engineeredtogether with an Fc domain such that the diabody molecule comprises ahinge-Fc domain.

In other embodiments, diabody molecules comprising Fc domains may betetramers, which may comprise two “heavier” polypeptide chains (i.e. apolypeptide chain comprising a V_(L), a V_(H) and an Fc domain), and two“lighter” polypeptide chains (i.e., a polypeptide chain comprising aV_(L) and a V_(H)). Such lighter and heavier chains may interact to forma monomer, and interact via their unpaired Fc domains to form an Ig-likemolecule, which may be a DART molecule. Such an Ig-like diabody istetravalent and may be monospecific, bispecific or tetraspecific. TheIg-like DART species has unique properties, because its domains may bedesigned to bind to the same epitope (so as to form a tetravalent,mono-epitope specific Ig-like DART capable of binding four identicalantigen molecules), or to different epitopes or antigens. For example,its domains may be designed to bind to two epitopes of the same antigen(so as to form a tetravalent, mono-antigen specific, bi-epitope specificIg-like DART), or to epitopes of different antigen molecules so as toform a tetravalent Ig-like DART having a pair of binding sites specificfor a first antigen and a second pair of binding sites specific for asecond antigen). Hybrid molecules having combinations of such attributescan be readily produced.

Although not intending to be bound by a particular mechanism of action,the diabody molecules of the invention exhibit enhanced therapeuticefficacy relative to therapeutic antibodies known in the art, in part,due to the ability of diabody to immunospecifically bind a target cellwhich expresses a particular antigen (e.g., FcγR) at reduced levels, forexample, by virtue of the ability of the diabody to remain on the targetcell longer due to an improved avidity of the diabody-epitopeinteraction. Thus, the diabodies of the invention have particularutility in treatment, prevention or management of a disease or disorder,such as cancer, in a sub-population, wherein the target antigen isexpressed at low levels in the target cell population.

Due to their increased valency, low dissociation rates and rapidclearance from the circulation (for diabodies of small size, at or below˜50 kDa), diabody molecules known in the art have also shown particularuse in the filed of tumor imaging. (Fitzgerald et al. (1997) ProteinEng. 10:1221). Of particular importance is the cross linking ofdiffering cells, for example the cross linking of cytotoxic T cells totumor cells. (Staerz et al. (1985) Nature 314:628-631; Holliger et al.(1996) Protein Eng. 9:299-305). Diabody epitope binding domains may alsobe directed to a surface determinant of any immune effector cell such asCD3, CD16, CD32, or CD64, which are expressed on T lymphocytes, naturalkiller (NK) cells or other mononuclear cells. In many studies, diabodybinding to effector cell determinants, e.g., Fcγ receptors (FcγR), wasalso found to activate the effector cell. (Holliger et al. (1996)Protein Eng. 9:299-305; Holliger et al. (1999) Cancer Res.59:2909-2916). Normally, effector cell activation is triggered by thebinding of an antigen bound antibody to an effector cell via Fc-FcγRinteraction; thus, in this regard, diabody molecules of the inventionmay exhibit Ig-like functionality independent of whether they comprisean Fc domain. By cross-linking tumor and effector cells, the diabody notonly brings the effector cell within the proximity of the tumor cellsbut leads to effective tumor killing Cao and Lam (2003) Adv. Drug.Deliv. Rev. 55:171-97.

The diabody molecules of the present invention can be produced using avariety of methods, including de novo protein synthesis and recombinantexpression of nucleic acids encoding the binding proteins. The desirednucleic acid sequences can be produced by recombinant methods (e.g., PCRmutagenesis of an earlier prepared variant of the desiredpolynucleotide) or by solid-phase DNA synthesis. Preferably recombinantexpression methods are used. In one aspect, the invention provides apolynucleotide that comprises a sequence encoding a CD16A V_(H) and/orV_(L); in another aspect, the invention provides a polynucleotide thatcomprises a sequence encoding a CD32B V_(H) and/or V_(L). Because of thedegeneracy of the genetic code, a variety of nucleic acid sequencesencode each immunoglobulin amino acid sequence, and the presentinvention includes all nucleic acids encoding the binding proteinsdescribed herein.

B7. Production of Antibodies

The antibodies of the preferred embodiments of the invention may beproduced or obtained in any of a variety of ways. For example, suchantibodies may be obtained from plasma, synthetically, recombinantly ortransgenically, via cell (e.g., hybridoma culture), etc. The productionof synthetic proteins has been described in, e.g., Dawson et al. (2000)Ann. Rev Biochem. 69:923-960; Wilken et al. (1998) Curr. Opin.Biotechnol. 9(4):412-426; and Kochendoerfer et al. (1999) Curr. Opin.Chem. Biol. 3(6):665-671.

Production of recombinant and transgenic antibodies has been describedin, e.g., Wang et al. (2007) IDrugs 10(8):562-565; Hagemeyer et al.(2007) Semin. Thromb. Hemost. 33(2):185-195; Rasmussen et al. (2007)Biotechnol. Lett. 29(6):845-852; Gasser et al. (2007) Biotechnol. Lett.29(2):201-212; Aubrey et al. (2006) J. Soc. Biol. 200(4):345-354; Lafflyet al. (2006) J. Soc. Biol. 200(4):325-343; Jefferis (2005) BiotechnolProg. 21(1):11-16; Smith et al. (2004) J. Clin. Pathol. 57(9):912-917;Kipriyanov et al. (2004) Mol Biotechnol. 26(1):39-60; Fischer et al.(2003) Vaccine 21(7-8):820-825; Maynard et al. (2000) Ann. Rev. Biomed.Eng. 2:339-376; Young et al. (1998) Res. Immunol. 149(6):609-610; andHudson (1998) Curr. Opin. Biotechnol. 9(4):395-402.

Production of antibodies via cell (e.g., hybridoma) culture has beendescribed in, e.g., Laffly et al. (2006), supra; Aldington et al. (2007)J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 848(1):64-78; S. S.Farid (2006) J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.848(1):8-18; Birch et al. (2006) Adv. Drug Deliv. Rev. 58(5-6):671-685;Even et al. (2006) Trends Biotechnol. 24(3):105-108; Graumann et al.(2006) Biotechnol. J. 1(2):164-86; U.S. Pat. No. 7,112,439; and U.S.Patent Publications Nos. 20070037216 and 20040197866.

Antibodies may be produced via phage display methods, such as thosedisclosed in, e.g., Brinkman et al. (1995) J. Immunol. Methods182:41-50; Ames et al. (1995) J. Immunol. Methods 184:177-86;Kettleborough et al. (1994) Eur. J. Immunol. 24:952-58; Persic et al.(1997) Gene 187:9-18; Burton et al. (1994) Advances in Immunology57:191-280; PCT Publications WO 90/02809; WO 91/10737; WO 92/01047; WO92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108. Phage display technology can also be used toincrease the affinity of an antibody for its antigen. The technology,referred to as affinity maturation, employs mutagenesis or CDR walkingand re-selection using the cognate antigen to identify antibodies thatbind with higher affinity to the antigen when compared with the initialor parental antibody. See, e.g., Glaser et al. (1992) J. Immunology149:3903; Wu et al. (1998) Proc. Natl. Acad. Sci. (U.S.A.) 95:6037;Yelton et al. (1995) J. Immunology 155:1994; Schier et al. (1996) J.Mol. Bio. 263:551.

Monoclonal antibodies may be made by a variety of methods known to thoseskilled in the art, for example, hybridoma methods as described in,e.g., Kohler et al. (1975) Nature 256:495, Kozbor et al. (1983)Immunology Today 4:72, or Cole et al. (1985) Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96, or recombinant DNAmethods as described in, e.g., U.S. Pat. No. 4,816,567, or theantibodies may be isolated from phage antibody libraries using thetechniques described in Clackson et al. (1991) Nature 352:624-628 andMarks et al. (1991) J. Mol. Biol. 222:581-597, for example Variousprocedures well known in the art may be used for the production ofpolyclonal antibodies to an antigen of interest. For example, varioushost animals can be immunized by injection with an antigen of interestor derivative thereof, including but not limited to rabbits, sheep,goats, dogs, mice, rats, and guinea pigs, and after allowing for animmunological response, the antibodies can be identified from the seraof the immunized animals.

Bispecific antibodies may also be made, for example through theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two chains have different specificities, followed by purification ofthe desired molecule using affinity chromatography, as described byMilstein et al. (1983) Nature 305:537-39, WO 93/08829, Traunecker et al.(1991) EMBO J. 10:3655-59. In a different approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences,for example to a heavy chain constant domain, comprising at least partof the hinge, C_(H2), and C_(H3) regions. The nucleic acids encodingthese fusions may be inserted into the same or different expressionvectors, and are expressed in a suitable host organism.

Fully human antibodies (also referred to as completely human antibodies)may be produced using transgenic mice that are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. An overview ofthis technology for producing human antibodies is described in, forexample, Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93, and U.S.Pat. No. 5,633,425. Fully human antibodies can also be produced usingother techniques known in the art, including phage display libraries, asdescribed by Hoogenboom and Winter (1991) J. Mol. Biol. 227:381 andMarks et al. (1991) J. Mol. Biol. 222:581. Fully human antibodies mayalso be obtained commercially from, for example, Abgenix, Inc.(Freemont, Calif.) and Genpharm (San Jose, Calif.). Fully humanantibodies that recognize a selected epitope may be generated using atechnique referred to as “guided selection.” In this approach a selectednon-human monoclonal antibody, e.g., a mouse antibody, is used to guidethe selection of a completely human antibody recognizing the sameepitope, as described by, e.g., Jespers et al. (1994) Biotechnology12:899-903.

The present invention also includes polynucleotides that encode themolecules of the invention, including the polypeptides and antibodies,as well as vectors comprising the polynucleotides, and host cellscomprising the vectors. The polynucleotides encoding the molecules ofthe invention may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art, for example,recombinant DNA techniques, site directed mutagenesis, PCR, etc. In oneembodiment, human libraries or any other libraries available in the art,can be screened by standard techniques known in the art, to clone thenucleic acids encoding the molecules of the invention.

B8. Characterization of Antibodies

The antibodies of the present invention may be characterized in avariety of ways. In particular, antibodies of the invention may beassayed for the ability to immunospecifically bind to an antigen, e.g.,HER2/neu, or, where the molecule comprises an Fc domain (or portionthereof) for the ability to exhibit Fc-FcγR interactions, i.e. specificbinding of an Fc domain (or portion thereof) to an FcγR. Such an assaymay be performed in solution (e.g., Houghten (1992) Bio/Techniques13:412-421), on beads (Lam (1991) Nature 354:82-84), on chips (Fodor(1993) Nature 364:555-556), on bacteria (U.S. Pat. No. 5,223,409), onspores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. (U.S.A.)87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310). Moleculesthat have been identified to immunospecifically bind to an antigen canthen be assayed for their specificity and affinity for the antigen.

Immunoassays which can be used to analyze immunospecific binding,cross-reactivity, and Fc-FcγR interactions include, but are not limitedto, competitive and non-competitive assay systems using techniques suchas western blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunochromatographicassays, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, and protein A immunoassays, etc. (see, e.g., Ausubel etal., 2008, Current Protocols in Molecular Biology).

Binding affinity for a target antigen is typically measured ordetermined by standard antibody-antigen assays, such as Biacorecompetitive assays, saturation assays, or immunoassays such as ELISA orRIA.

Preferably, fluorescence activated cell sorting (FACS), using any of thetechniques known to those skilled in the art, is used for immunologicalor functional based assays to characterize molecules of the invention.Flow sorters are capable of rapidly examining a large number ofindividual cells that have been bound, e.g., opsonized, by molecules ofthe invention (e.g., 10-100 million cells per hour). Additionally,specific parameters used for optimization of antibody behavior, includebut are not limited to, antigen concentration, kinetic competition time,or FACS stringency, each of which may be varied in order to select forantibody molecules which exhibit specific binding properties. Flowcytometers for sorting and examining biological cells are well known inthe art. Known flow cytometers are described, for example, in U.S. Pat.Nos. 4,347,935; 5,464,581; 5,483,469; 5,602,039; 5,643,796; and6,211,477. Other known flow cytometers are the FACS Vantage™ system soldby Becton Dickinson and Company, and the COPAS™ system sold by UnionBiometrica.

Surface plasmon resonance-based assays may be used to characterize thekinetic parameters of an antigen-binding domain or Fc-FcγR binding. Anymethod known to those skilled in the art may be used, for example thetechnology described in, e.g., Dong et al. (2002) Review in Mol.Biotech. 82:303-323; Mullet et al. (2000) Methods 22:77-91; Rich et al.(2000) Current Opinion in Biotechnology 11:54-61; Fivash et al. (1998)Current Opinion in Biotechnology 9:97-101; and U.S. Pat. Nos. 6,373,577;6,289,286; 5,322,798; 5,341,215; and 6,268,125. The data is used to plotbinding curves and determine rate constants, for example, K_(on),K_(off), and the apparent equilibrium binding constant K_(d), forexample as described in, e.g., Myszka (1997) Current Opinion inBiotechnology 8:50-57; O'Shannessy et al. (1996) Analytical Biochemistry236:275-283; Morton et al. (1995) Analytical Biochemistry 227:176-185;Fisher et al. (1994) Current Opinion in Biotechnology 5:389-95;O'Shannessy (1994) Current Opinion in Biotechnology 5:65-71; and Chaikenet al. (1992) Analytical Biochemistry 201:197-210. In preferredembodiments, the kinetic parameters determined using an SPR analysis maybe used as a predictive measure of how a molecule will function in afunctional assay, e.g., ADCC.

Characterization of binding to FcγR by molecules comprising an Fc domain(or portion thereof) and/or comprising epitope binding domain specificfor an FcγR may be performed according to the methods described in theAntibody Engineering Technology Art. Assays for effector cell functionsare well-known, for example as described in Abdul-Majid et al. (2002)Scand. J. Immunol. 55:70-81; Perussia et al. (2000) Methods Mol. Biol.121:179-192; Lehmann et al. (2000) J. Immunol. Methods 243(1-2):229-242;Ding et al. (1998) Immunity 8:403-411; Baggiolini et al. (1998)Experientia 44(10):841-848; Brown (1994) Methods Cell Biol. 45:147-164;and Munn et al. (1990) J. Exp. Med. 172:231-237.

For example, assays for FcγR-mediated phagocytosis may be conductedusing human monocytes, by measuring the ability of THP-1 cells tophagocytose fluoresceinated IgG-opsonized sheep red blood cells (SRBC)by methods previously described in Tridandapani et al. (2000) J. Biol.Chem. 275:20480-20487, or using an antibody-dependent opsonophagocytosisassay (ADCP) as described by Bedzyk et al. (1989) J. Biol. Chem.264(3):1565-1569. Standard methods known to those skilled in the art maybe used to characterize the binding of C1q and mediation of complementdependent cytotoxicity (CDC) by molecules of the invention comprising Fcdomains (or portions thereof). For example, to determine C1q binding, aC1q binding ELISA may be performed, and to assess complement activation,a complement dependent cytotoxicity (CDC) assay may be performed, e.g.,as described in Gazzano-Santoro et al. (1996) J. Immunol. Methods202:163.

In another embodiment, the molecules of the invention can be assayed forFcγR-mediated ADCC activity in effector cells, e.g., natural killercells, using any of the standard methods known to those skilled in theart and described in, e.g., Weng et al. (2003) J. Clin. Oncol.21:3940-3947; Perussia et al. (2000) Methods Mol. Biol. 121:179-192;Ding et al. (1998) Immunity 8:403-411. In a specific preferredembodiment, a time resolved fluorimetric assay is used for measuringADCC activity against fluorescently-labeled target cells, as describedin, e.g., Blomberg et al. (1996) Journal of Immunological Methods193:199-206. Target cells used in the ADCC assays of the inventioninclude, but are not limited to, breast cancer cell lines, e.g., SK-BR-3with ATCC accession number HTB-30 (Tremp et al. (1976) Cancer Res.33-41); B-lymphocytes; cells derived from Burkitts lymphoma, e.g., Rajicells with ATCC accession number CCL-86 (Epstein et al. (1965) J. Natl.Cancer Inst. 34:231-240), and Daudi cells with ATCC accession numberCCL-213 (Klein et al. (1968) Cancer Res. 28:1300-1310). The target cellsmust be recognized by the antigen binding site of the molecule to beassayed. Preferably, the effector cells used in the ADCC assays of theinvention are peripheral blood mononuclear cells (PBMC) that arepreferably purified from normal human blood, using standard methodsknown to one skilled in the art, e.g., using Ficoll-Paque densitygradient centrifugation.

C. Methods of Treatment & Pharmaceutical Compositions

The administration of the compositions (e.g., antibodies andpolypeptides) of the present invention may be for a “prophylactic” or“therapeutic” purpose, or alternatively can be used for diagnosticpurposes. The compositions of the present invention are said to beadministered for a “therapeutic” purpose if the amount administered isphysiologically significant to provide a therapy for an actualmanifestation of the disease. When provided therapeutically, thecompound is preferably provided at (or shortly after) the identificationof a symptom of actual disease. The therapeutic administration of thecompound serves to attenuate the severity of such disease or to reverseits progress. The compositions of the present invention are said to beadministered for a “prophylactic” purpose if the amount administered isphysiologically significant to provide a therapy for a potential diseaseor condition. When provided prophylactically, the compound is preferablyprovided in advance of any symptom thereof. The prophylacticadministration of the compound serves to prevent or attenuate anysubsequent advance or recurrence of the disease.

Providing a therapy or “treating” refers to any indicia of success inthe treatment or amelioration of an injury, pathology or condition,including any objective or subjective parameter such as abatement,remission, diminishing of symptoms or making the injury, pathology orcondition more tolerable to the patient, slowing in the rate ofdegeneration or decline, making the final point of degeneration lessdebilitating, or improving a patient's physical or mental well-being.The treatment or amelioration of symptoms can be based on objective orsubjective parameters, including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation.

Preferred subjects for treatment include animals, most preferablymammalian species such as humans or other primates, and domestic animalssuch as dogs, cats and the like, subject to disease and otherpathological conditions. A “patient” refers to a subject, preferablymammalian (including human).

Certain embodiments of the present invention relate to pharmaceuticalcompositions comprising one or more therapeutic agents, and methods ofadministering a therapeutically effective amount of one or moretherapeutic agents, which are capable of prophylactic and/or therapeutictreatment of disorders. The term “therapeutic agent” refers to any agenthaving a therapeutic effect to prophylactically or therapeutically treata disorder. Exemplary therapeutic agents include the antibodies andpolypeptides of the present invention, as well as other therapeuticagents that may be administered in combination with, or conjugated to,an antibody or polypeptide. In a preferred embodiment, the therapeuticagent is an antibody of the present invention, and preferably is anantibody fragment, a diabody, an Ig-like DART, or a fusion protein.

The molecules of the invention are particularly useful for the treatmentand/or prevention of a disease, disorder or infection where an effectorcell function (e.g., ADCC) mediated by FcγR is desired (e.g., cancer,infectious disease). For example, molecules of the invention may bind acell surface antigen and an FcγR (e.g., FcγRIIIA) on an immune effectorcell (e.g., NK cell), stimulating an effector function (e.g., ADCC, CDC,phagocytosis, opsonization, etc.) against said cell. In someembodiments, the antibodies and polypeptides of the invention areespecially suited for the treatment of cancers. The efficacy of standardmonoclonal antibody therapy depends on the FcγR polymorphism of thesubject. Carton et al. (2002) Blood 99:754-758; Weng et al. (2003) JClin Oncol. 21(21):3940-3947. These receptors are expressed on thesurface of the effector cells and mediate ADCC. High affinity allelesimprove the effector cells' ability to mediate ADCC. The antibodies andpolypeptides of the invention may comprise a variant Fc domain thatexhibits enhanced affinity to FcγR (relative to a wild type Fc domain)on effector cells, thus providing better immunotherapy reagents forpatients regardless of their FcγR polymorphism.

For diagnostic purposes, the antibodies or polypeptides may be coupledto a detectable substance, so that they can be used, for example, tomonitor the development or progression of a disease, disorder orinfection. Examples of detectable substances include various enzymes(e.g., horseradish peroxidase, beta-galactosidase, etc.), prostheticgroups (e.g., avidin/biotin), fluorescent materials (e.g.,umbelliferone, fluorescein, or phycoerythrin), luminescent materials(e.g., luminol), bioluminescent materials (e.g., luciferase oraequorin), radioactive materials (e.g., carbon-14, manganese-54,strontium-85 or zinc-65), positron emitting metals, and nonradioactiveparamagnetic metal ions. The detectable substance may be coupled orconjugated either directly to the molecules of the invention orindirectly through an intermediate (e.g., a linker), using techniquesknown in the art.

C1. Treatable Disorders

Exemplary disorders that may be treated by various embodiments of thepresent invention include, but are not limited to, proliferativedisorders, cell proliferative disorders, and cancer, autoimmunediseases, inflammatory disorders, and infectious diseases. In variousembodiments, the invention encompasses methods and compositions fortreatment, prevention or management of a disease or disorder in asubject, comprising administering to the subject a therapeuticallyeffective amount of one or more molecules (antibodies or polypeptides)which bind to a disease antigen. For example, molecules of the inventionare particularly useful for the prevention, inhibition, reduction ofgrowth or regression of primary tumors, metastasis of cancer cells, andinfectious diseases. Although not intending to be bound by a particularmechanism of action, molecules of the invention mediate effectorfunction resulting in tumor clearance, tumor reduction or a combinationthereof. In alternate embodiments, diabodies of the invention mediatetherapeutic activity by cross-linking of cell surface antigens and/orreceptors and enhanced apoptosis or negative growth regulatorysignaling.

Antibodies with a decreased affinity for FcγRIIB and an increasedaffinity for FcγRIIIA and/or FcγRIIA may lead to an enhanced activatingresponse upon FcγR binding and thus have therapeutic efficacy fortreating and/or preventing cancer. Non-limiting examples of cancerstreatable by the methods herein include acute myeloid lymphoma, adrenalcarcinoma, adenocarcinoma, basal cancer, bladder cancer, bone cancer,bone and connective tissue sarcoma, brain cancer, breast cancer,bronchial cancer, cervical cancer, choriocarcinoma, chronic lymphocyticleukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer,endometrial cancer, esophageal cancer, eye cancer, fallopian tubecancer, gall bladder cancer, gastrointestinal cancer, glioma, hairy cellleukemia, hepatoma, Hodgkin's disease, intrahepatic bile duct cancer,joint cancer, Kaposi's sarcoma, kidney cancer, larynx cancer, livercancer, leukemia, lung cancer, lymphoblastic leukemia, lymphoma,malignant mesothelioma, medullobastoma, melanoma, mesothelioma, middleear cancer, multiple myeloma, myeloma, myxosarcoma, nasal cavity cancer,nasopharynx cancer, neuroblastoma, Non-Hodgkin's lymphoma, non-smallcell lung cancer, nose cancer, oral cavity cancer, ovarian cancer,pancreatic cancer, penal cancer, peritoneum cancer, pharynx cancer,pituitary gland cancer, prostate cancer, rectal cancer, renal cancer,salivary gland cancer, skin cancer, soft tissue sarcoma, squamous cellcarcinoma, stomach cancer, testicular cancer, thyroid cancer, urinarycancer, uterine cancer, vaginal cancer, vesticular cancer, vulvalcancer, and Wilm's tumor.

In some embodiments, the cancer is a hematopoietic cancer orblood-related cancer, such as lymphoma, leukemia, myeloma, lymphoidmalignancy, cancer of the spleen, and cancer of the lymph nodes. In apreferred embodiment, the cancer is a B-cell associated cancer, such as,for example, high, intermediate or low grade lymphoma (including B celllymphoma such as, for example, Burkitt's lymphoma, diffuse large celllymphoma, follicular lymphoma, Hodgkin's lymphoma, mantle cell lymphoma,marginal zone lymphoma, mucosa-associated-lymphoid tissue B celllymphoma, non-Hodgkin's lymphoma, small lymphocytic lymphoma, and T celllymphomas) and leukemias (including chronic lymphocytic leukemia, suchas B cell leukemia (CD5+ B lymphocytes), chronic myeloid leukemia,lymphoid leukemia, such as acute lymphoblastic leukemia, myelodysplasia,myeloid leukemia, such as acute myeloid leukemia, and secondaryleukemia), multiple myeloma, such as plasma cell malignancy, and otherhematological and/or B cell- or T-cell-associated cancers. Otherexemplary cancers are cancers of additional hematopoietic cells,including polymorphonuclear leukocytes, such as basophils, eosinophils,neutrophils and monocytes, dendritic cells, platelets, erythrocytes andnatural killer cells.

In some embodiments, the cancer to be treated is breast cancer, prostatecancer, uterine cancer, ovarian cancer, colon cancer, endometrialcancer, adrenal carcinoma, or non-small cell lung cancer. In someembodiments, the cancer is breast cancer or prostate cancer. In someembodiments, the cancer is a cancer in which HER2/neu is overexpressed.In a specific embodiment, an antibody or polypeptide of the inventioninhibits or reduces the growth of cancer cells by at least 99%, at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, at least 50%, at least 45%, at least 40%, at least45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least10% relative to the growth of cancer cells in the absence of theantibody or polypeptide of the invention.

Antibodies with an increased affinity for FcγRIIB and a decreasedaffinity for FcγRIIIA and/or FcγRIIA may lead to a diminished activatingresponse upon FcγR binding and thus have therapeutic efficacy fortreating and/or preventing inflammation and autoimmune disease. Examplesof autoimmune disorders that may be treated by the methods hereininclude, but are not limited to, alopecia areata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy,celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis,Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathicpulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgAneuropathy, juvenile arthritis, lichen planus, lupus erythematosus,Ménière's disease, mixed connective tissue disease, multiple sclerosis,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, takayasu arteritis, temporalarteristis/giant cell arteritis, type 1 or immune-mediated diabetesmellitus, ulcerative colitis, uveitis, vasculitides such as dermatitisherpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.

Non-limiting examples of inflammatory disorders treatable by the methodsherein include immune-mediated inflammatory disorders (IMIDs), which areinflammatory conditions caused and sustained by an antigen-specific,pathological immune response. Among these disorders are various types ofallergic diseases, such as asthma, hay fever, and urticaria, arthritis,such as osteoarthritis and rheumatoid arthritis, chronic inflammation,chronic obstructive pulmonary disease (COPD), connective tissuedisorders, fibrosis, graft rejection and graft-versus host-disease,inflammatory bowel disease (e.g., Crohn's disease and ulcerativecolitis), inflammatory osteolysis, insulin-dependent diabetes, pulmonaryfibrosis, retinitis, undifferentiated arthropathy, undifferentitatedspondyloarthropathy, and uveitis. Molecules of the invention comprisingat least one epitope binding domain specific for FcγRIIB and/or avariant Fc domain with an enhanced affinity for FcγRIIB and a decreasedaffinity for FcγRIIIA can also be used to prevent the rejection oftransplants.

The anti-inflammatory polypeptides of the present invention willpreferably reduce inflammation in an animal by at least 99%, at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, at least 50%, at least 45%, at least 40%, at least45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least10% relative to the inflammation in an animal that does not receive suchpolypeptides.

In certain embodiments, the polypeptides of the invention are toxic toan infectious agent, enhance immune response against said agent orenhance effector function against said agent, relative to the immuneresponse in the absence of said molecule. Infectious diseases that canbe treated or prevented by the molecules of the invention are caused byinfectious agents including but not limited to bacteria, fungi,protozoans, and viruses. Non-limiting exemplary bacterial diseasesinclude those caused by Bacillus antracis (anthrax), Borreliaburgdorferi (Lyme disease), Candida, chlamydia, cholera, diptheria, E.coli, Enterococcus faecials, Heliobacter pylori, Klebsiella pneumoniae,legionella, mycobacterium, mycoplasma, Neisseria, pertussis, plague,Proteus vulgaris, Pseudomonas aeruginosa, S. pneumonia, Salmonella,staphylococcus, streptococcus, and tetanus. Non-limiting protozoaldiseases include those caused by kokzidioa, leishmania, malaria, ortrypanosoma.

Non-limiting examples of viral diseases include those caused byadenovirus, arbovirus, coronavirus, coxsackie virus, cytomegalovirus,ebola, echinovirus, echovirus, endotoxin (LPS), enterovirus, EpsteinBarr virus, hepatitis virus (e.g., hepatitis type A, hepatitis type B,hepatitis type C, murine hepatitis), herpes virus (e.g., herpes simplextype I (HSV-I), herpes simplex type II (HSV-II), murine gamma herpesvirus), human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), huntavirus, influenza, leukemiavirus (e.g., murine leukemia, feline leukemia, etc.); measles virus,mumps virus, papilloma virus, papova virus, polio virus, respiratorysyncytial virus, retrovirus, rhinovirus, rinderpest, rotavirus, rubellavirus, small pox, T-cell lymphotropic virus 1, vaccinia, varicella, andagents of viral diseases such as viral meningitis, encephalitis, ordengue.

C2. Formulations

The pharmaceutical compositions can be formulated according to knownmethods for preparing pharmaceutically useful compositions, and mayinclude a pharmaceutically acceptable carrier and/or an excipient. Thecompositions can be in any suitable form, for example tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing, for example, up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders, to name just a fewnon-limiting alternatives. Such compositions may be prepared by anyknown method, for example by admixing the active ingredient with thecarrier(s) or excipient(s) under sterile conditions.

The active ingredients can also be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.The physical and chemical characteristics of the compositions of theinvention may be modified or optimized according to the skill in theart, depending on the mode of administration and the particular diseaseor disorder to be treated. The compositions may be provided in unitdosage form, a sealed container, or as part of a kit, which may includeinstructions for use and/or a plurality of unit dosage forms.

In particular embodiments, the therapeutic agents can be incorporatedinto a composition, by, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe antibody or fusion protein, receptor-mediated endocytosis (See,e.g., Wu and Wu (1987) J. Biol. Chem. 262:4429-4432), construction of anucleic acid as part of a retroviral or other vector, etc. In anotherparticular embodiment, the therapeutic agents are supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted, e.g., with wateror saline to the appropriate concentration for administration to asubject.

Preferably, the therapeutic agent is supplied as a dry sterilelyophilized powder in a hermetically sealed container at a unit dosageof at least 5 mg, more preferably at least 10 mg, at least 15 mg, atleast 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least75 mg. The lyophilized powder should be stored at between 2 and 8° C. inits original container and the molecules should be parenterallyadministered within 12 hours, preferably within 6 hours, within 5 hours,within 3 hours, or within 1 hour after being reconstituted. In analternative embodiment, the therapeutic agents are supplied in liquidform in a hermetically sealed container indicating the quantity andconcentration of the therapeutic agent. Preferably, the liquid form issupplied in a hermetically sealed container at least 1 mg/ml, morepreferably at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, atleast 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml,at least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml of themolecules.

C3. Kits

The compositions may also be included in a kit. The kit can include, innon-limiting aspects, a pharmaceutical composition comprising atherapeutic agent, instructions for administration and/or othercomponents. In preferred embodiments, the kit can include a compositionready for administration. Containers of the kits can include a bottle,dispenser, package, compartment, or other types of containers, intowhich a component may be placed. The container can include indicia onits surface. The indicia, for example, can be a word, a phrase, anabbreviation, a picture, or a symbol. The containers can dispense apre-determined amount of the component (e.g. compositions of the presentinvention). The composition can be dispensed in a spray, an aerosol, orin a liquid form or semi-solid form. The containers can have spray,pump, or squeeze mechanisms. In certain aspects, the kit can include asyringe for administering the compositions of the present invention.

Where there is more than one component in the kit (they may be packagedtogether), the kit also will generally contain a second, third or otheradditional containers into which the additional components may beseparately placed. The kits of the present invention also can include acontainer housing the components in close confinement for commercialsale. Such containers may include injection or blow-molded plasticcontainers into which the desired bottles, dispensers, or packages areretained. A kit can also include instructions for employing the kitcomponents as well the use of any other compositions, compounds, agents,active ingredients, or objects not included in the kit. Instructions mayinclude variations that can be implemented. The instructions can includean explanation of how to apply, use, and maintain the products orcompositions, for example.

C4. Administration and Dosage

A variety of administration routes for the compositions of the presentinvention are available. The particular mode selected will depend, ofcourse, upon the particular therapeutic agent selected, whether theadministration is for prevention, diagnosis, or treatment of disease,the severity of the medical disorder being treated and dosage requiredfor therapeutic efficacy. The methods of this invention may be practicedusing any mode of administration that is medically acceptable, andproduces effective levels of the active compounds without causingclinically unacceptable adverse effects. Such modes of administrationinclude, but are not limited to, oral, buccal, sublingual, inhalation,mucosal, rectal, intranasal, topical, ocular, periocular, intraocular,transdermal, subcutaneous, intra-arterial, intravenous, intramuscular,parenteral, or infusion methodologies. In a specific embodiment, it maybe desirable to administer the pharmaceutical compositions of theinvention locally to the area in need of treatment; this may be achievedby, for example, and not by way of limitation, local infusion, byinjection, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,healing or amelioration of chronic conditions, a reduction in symptoms,an increase in rate of healing of such conditions, or a detectablechange in the levels of a substance in the treated or surroundingtissue. When applied to an individual active ingredient, administeredalone, the term refers to that ingredient alone. When applied to acombination, the term refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially, or simultaneously.

The dosage schedule and amounts effective for therapeutic andprophylactic uses, i.e., the “dosing regimen”, will depend upon avariety of factors, including the stage of the disease or condition, theseverity of the disease or condition, the general state of the patient'shealth, the patient's physical status, age and the like. Therapeuticefficacy and toxicity of the compositions may be determined by standardpharmaceutical, pharmacological, and toxicological procedures in cellcultures or experimental animals. For example, numerous methods ofdetermining ED₅₀ (the dose therapeutically effective in 50 percent ofthe population) and LD₅₀ (the dose lethal of 50 percent of thepopulation) exist. The dose ratio between therapeutic and toxic effectsis the therapeutic index, and it can be expressed as the ratioED₅₀/LD₅₀. Compositions exhibiting high therapeutic indices arepreferred. The data obtained from cell culture assays or animal studiesmay be used in formulating a range of dosages for human use. The dosageis preferably within a range of concentrations that includes the ED₅₀with little or no toxicity, and may vary within this range depending onthe dosage form employed, sensitivity of the patient, and the route ofadministration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the rate of absorption,bioavailability, metabolism, clearance, and the like (see, e.g.,Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;the latest Remington, supra). The state of the art allows the clinicianto determine the dosage regimen for each individual patient, therapeuticagent and disease or condition treated. Single or multipleadministrations of the compositions of the present invention can beadministered depending on the dosage and frequency as required andtolerated by the patient. The duration of prophylactic and therapeutictreatment will vary depending on the particular disease or conditionbeing treated. Some diseases lend themselves to acute treatment whereasothers require long-term therapy. If administration is not on a dailybasis, for example if injections are given every few days, every fewweeks, or every few months, then more therapeutic agent may be includedin each administration, so that daily release of the agent is adequateto meet therapeutic needs.

In a preferred embodiment, the therapeutic agents of the invention areadministered in metronomic dosing regimens, either by continuousinfusion or frequent administration without extended rest periods. Suchmetronomic administration can involve dosing at constant intervalswithout rest periods. Typically the therapeutic agents, in particularcytotoxic agents, are used at lower doses. Such dosing regimensencompass the chronic daily administration of relatively low doses forextended periods of time, which can minimize toxic side effects andeliminate rest periods. Kamat et al. (2007) Cancer Research 67:281-88.In certain embodiments, the therapeutic agents are delivered by chroniclow-dose or continuous infusion ranging from about 24 hours to about 2days, to about 1 week, to about 2 weeks, to about 3 weeks to about 1month to about 2 months, to about 3 months, to about 4 months, to about5 months, to about 6 months. The scheduling of such dose regimens can beoptimized by the skilled oncologist.

For antibodies encompassed by the invention, the dosage administered toa patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kgor 0.01 to 0.10 mg/kg of the patient's body weight. The dosage andfrequency of administration may be reduced or altered by enhancinguptake and tissue penetration of the antibodies by modifications suchas, for example, lipidation. In one embodiment, the dosage of theantibodies administered to a patient are 0.01 mg to 1000 mg/day, whenused as single agent therapy. In another embodiment the antibodies areused in combination with other therapeutic compositions and the dosageadministered to a patient are lower than when said molecules are used asa single agent therapy. In a preferred example, a subject is treatedwith antibodies in the range of between about 0.1 to 30 mg/kg bodyweight, one time per week for between about 1 to 10 weeks, preferablybetween 2 to 8 weeks, more preferably between about 3 to 7 weeks, andeven more preferably for about 4, 5, or 6 weeks.

C5. Combination Therapies

The invention further encompasses administering the antibodies orpolypeptides of the invention in combination with other therapies knownto those skilled in the art for the treatment or prevention of cancer,autoimmune disease, inflammation, or infectious disease, including butnot limited to, current standard and experimental chemotherapies,hormonal therapies, biological therapies, immunotherapies, radiationtherapies, or surgery. In some embodiments, the antibodies orpolypeptides of the invention may be administered in combination with atherapeutically or prophylactically effective amount of one or moretherapeutic agents known to those skilled in the art for the treatmentand/or prevention of cancer, autoimmune disease, infectious disease orintoxication.

As used herein, the term “combination” refers to the use of more thanone therapeutic agent. The use of the term “combination” does notrestrict the order in which therapeutic agents are administered to asubject with a disorder, nor does it mean that the agents areadministered at exactly the same time, but rather it is meant that anantibody or polypeptide of the invention and the other agent areadministered to a mammal in a sequence and within a time interval suchthat the antibody or polypeptide of the invention can act together withthe other agent to provide an increased benefit than if they wereadministered otherwise. For example, each therapeutic agent (e.g.,chemotherapy, radiation therapy, hormonal therapy or biological therapy)may be administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic or prophylactic effect. Each therapeutic agentcan be administered separately, in any appropriate form and by anysuitable route, e.g., one by the oral route and one parenterally.

In various embodiments, a first therapeutic agent can be administeredprior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second (or subsequent) therapeutic agent to asubject with a disorder. In preferred embodiments, two or more agentsare administered within the same patient visit, or no more than 12 hoursapart, no more than 24 hours apart, or no more than 48 hours apart.

In certain embodiments, the therapeutic agents are cyclicallyadministered to a subject. Cycling therapy involves the administrationof a first agent for a period of time, followed by the administration ofa second agent and/or third agent for a period of time and repeatingthis sequential administration. Cycling therapy can reduce thedevelopment of resistance to one or more of the therapies, avoid orreduce the side effects of one of the therapies, and/or improves theefficacy of the treatment. Exemplary cycles are about once every week,about once every 10 days, about once every two weeks, and about onceevery three weeks. Each cycle can comprise at least 1 week of rest, atleast 2 weeks of rest, at least 3 weeks of rest. The number of cyclesadministered is from about 1 to about 12 cycles, more typically fromabout 2 to about 10 cycles, and more typically from about 2 to about 8cycles.

In an embodiment for the treatment of a cell proliferative disorder, anantibody or polypeptide of the present invention is conjugated to, oradministered in combination with, another therapeutic agent, such as,but not limited to, an alkylating agent (e.g., mechlorethamine orcisplatin), angiogenesis inhibitor, anthracycline (e.g.,daunorubicin/daunomycin or doxorubicin), antibiotic (e.g., dactinomycin,bleomycin, or anthramycin), antibody (e.g., an anti-VEGF antibody suchas bevacizumab (sold as AVASTIN® by Genentech, Inc.), an anti-EGFRantibody such as panitumumab (sold as VECTIBIX™ by Amgen, Inc.), or ananti-integrin antibody such as natalizumab (sold as TYSABRI® by BiogenIdec and Elan Pharmaceuticals, Inc.)), an antimetabolite (e.g.,methotrexate or 5-fluorouracil), an anti-mitotic agent (e.g.,vincristine or paclitaxel), a cytotoxin (e.g., a cytostatic or cytocidalagent), a hormone therapy agent (e.g., a selective estrogen receptormodulator (e.g., tamoxifen or raloxifene), aromatase inhibitor,luteinizing hormone-releasing hormone analogue, progestational agent,adrenocorticosteroid, estrogen, androgen, anti-estrogen agent, androgenreceptor blocking agent, 5-alpha reductase inhibitor, adrenal productioninhibitor, etc.), a matrix metalloprotease inhibitor, a radioactiveelement (e.g., alpha-emitters, gamma-emitters, etc.), or any otherchemotherapeutic agent.

Non-limiting examples of suitable angiogenesis inhibitors includeABT-627; angiostatin (plasminogen fragment); angiozyme; antiangiogenicantithrombin III; Bay 12-9566; benefin; bevacizumab; BMS-275291;bisphosphonates; cartilage-derived inhibitor (CDI); CAI; CD59 complementfragment; CEP-7055; Col 3; combretastatin A-4; endostatin (collagenXVIII fragment); farnesyl transferase inhibitors (FTI); fibronectinfragment; gro-beta; halofuginone; heparinases; heparin hexasaccharidefragment; HMV833; human chorionic gonadotropin (hCG); IM-862; interferonalpha/beta/gamma; interferon inducible protein (IP-10); interleukin-12;kringle 5 (plasminogen fragment); marimastat; metalloproteinaseinhibitors (TIMPs); 2-methoxyestradiol; MMI 270 (CGS 27023A); MoAbIMC-1C11; neovastat; NM-3; panzem; PI-88; placental ribonucleaseinhibitor; plasminogen activator inhibitor; platelet factor-4 (PF4);prinomastat; prolactin 16 kDa fragment; proliferin-related protein(PRP); PTK 787/ZK 222594; retinoids; solimastat; squalamine; SS 3304; SU5416; SU6668; SU11248; tetrahydrocortisol-S; tetrathiomolybdate;thalidomide; thrombospondin-1 (TSP-1); TNP-470; transforming growthfactor-beta (TGF-b); vasculostatin; vasostatin (calreticulin fragment);ZD6126; and ZD 6474.

Non-limiting examples of additional antibodies for the treatment of acell proliferative disorder include antibodies to 17-1A, αvβ₃, AFP, CD3,CD18, CD20, CD22, CD33, CD44, CD52, CEA, CTLA-4, DNA-associatedproteins, EGF receptor, Ep-CAM, GD2-ganglioside, gp IIIb/IIIa, gp72,HER2, HLA-DR 10 beta, HLA-DR antigen, IgE, ganglioside GD3, MUC-1,nuC242, PEM antigen, SK-1 antigen, tumor antigen CA125, tumor antigenMUC1, VEGF, and VEGF-receptor.

In a different embodiment, an antibody or polypeptide of the presentinvention may be administered in combination with a therapeutic agent oragents for the treatment of an inflammatory disorder, such as, but notlimited to, antibodies, anticholingeric agents, beta-agonists, methylxanthines, non-steroidal anti-inflammatory drugs (NSAIDs) (e.g.,aspirin, ibuprofen, celecoxib or diclofenac), and steroidalanti-inflammatory drugs (e.g., glucocorticoids, dexamethasone,cortisones, prednisone or eicosanoids). The additional antibodies may beany suitable antibody for the treatment of inflammatory disease, suchas, but not limited to antibodies to alpha4beta7, beta2-integrin, CBL,CD2, CD3, CD4, CD11a, CD11/18, CD14, CD18, CD23, CD25, CD40L, CD64(FcR), CD80, CD147, Complement (C5), E-selectin, Fact VII, gpIIbIIIa,ICAM-3, IgE, IL-4, IL-5, IL-8, TNF-alpha, and VLA-4.

In a further embodiment, an antibody or polypeptide of the presentinvention may be administered in combination with a therapeutic agent oragents for the treatment of an autoimmune disorder, such as, but notlimited to, antibodies, brequinar, cyclophosphamide, cyclosporine A,cytokine receptor modulators, deoxyspergualin, leflunomide, macrolideantibiotics, malononitriloamindes (e.g., leflunamide), methothrexate,methylprednisolone, mizoribine, mycophenolate mofetil, rapamycin(sirolimus), steroids, and T cell receptor modulators. The additionalantibodies may be any suitable antibody for the treatment of anautoimmune disorder, and non-limiting examples include antibodies toa4b7 integrin receptor, CBL antigen, CD2, CD4, CD23, CD40, CD80, FcRI,Gamma Interferon, IL-8, inosine monophosphate dehydrogenase, ICEinterleukin-1 beta, P38MAP kinase, and TNF.

In still another embodiment, an antibody or polypeptide of the presentinvention may be administered in combination with a therapeutic agent oragents for the treatment of an infectious disease, such as, but notlimited to, an antibiotic, anti-fungal, or anti-viral agent. Antibioticsthat can be used in combination with the molecules of the inventioninclude, but are not limited to, 2,4 diaminopyrimidines (e.g.,brodimoprim), aminoglycosides (e.g., apramycin, neomycin, orspectinomycin), amphenicols (e.g., chloramphenicol), amphomycins,ansamycins (e.g., rifamide and rifampin), bacitracins, carbacephems(e.g., loracarbef), carbapenems (e.g., biapenem and imipenem),cephalosporins (e.g., cephalexin or cefadroxil), cephamycins (e.g.,cefbuperazone, cefmetazole, and cefminox), clarithromycins,erythromycins, lincosamides (e.g., clindamycin and lincomycin),macrolides (e.g., tobramycin), monobactams (e.g., carumonam),nitrofurans (e.g., furaltadone, and furazolium chloride), oxacephems(e.g., flomoxef and moxalactam), penicillins, quinolones (e.g.,ofloxacin or ciprofloxacin), sulfonamides (e.g., benzylsulfamide, andsulfacytine), sulfones (e.g., diathymosulfone, glucosulfone sodium, andsolasulfone), and tetracyclines (e.g., apicycline andchlortetracycline).

Antifungal agents that can be used in combination with the molecules ofthe invention include, but are not limited to, amphotericin B,butoconazole, ciclopirox, clotrimazole, econazole, fluconazole,flucytosine, griseofuldin, haloprogrin, intrathecal, itraconazole,ketoconazole, miconazole, naftifine, nystatin, terbinafine, terconazole,tioconazole, and undecylenate. Useful anti-viral agents that can be usedin combination with the molecules of the invention include, but are notlimited to, non-nucleoside reverse transcriptase inhibitors, nucleosideanalogs, nucleoside reverse transcriptase inhibitors, and proteaseinhibitors. Non-limiting examples of such agents are acyclovir,adefovir, alpha interferons, amantadine, amprenavir, clevadine,entecavir, foscarnet, gangcyclovir, idoxuridine, indinavir, lopinavir,pleconaril, ribavirin, rimantadine, ritonavir, saquinavir, trifluridine,vidarabine, and zidovudine.

C6. Demonstration of Therapeutic Utility

The pharmaceutical compositions, prophylactic, or therapeutic agents ofthe invention are preferably tested in vitro, in a cell culture system,and in an animal model organism, such as a rodent animal model system,for the desired therapeutic activity prior to use in humans. Forexample, assays which can be used to determine whether administration ofa specific pharmaceutical composition is desired, include cell cultureassays in which a patient tissue sample is grown in culture, and exposedto or otherwise contacted with a pharmaceutical composition of theinvention, and the effect of such composition upon the tissue sample isobserved. The tissue sample can be obtained by biopsy from the patient.This test allows the identification of the therapeutically mosteffective prophylactic or therapeutic molecule(s) for each individualpatient. In various specific embodiments, in vitro assays can be carriedout with representative cells of cell types involved in an autoimmune orinflammatory disorder (e.g., T cells), to determine if a pharmaceuticalcomposition of the invention has a desired effect upon such cell types.

Suitable animal model systems include, but are not limited to, rats,mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animalsystem well-known in the art may be used. In a specific embodiment ofthe invention, combinations of prophylactic and/or therapeutic agentsare tested in a mouse model system. Preferred animal models for use inthe methods of the invention are, for example, transgenic miceexpressing human FcγRs on mouse effector cells, e.g., any mouse modeldescribed in U.S. Pat. No. 5,877,396 can be used in the presentinvention.

Anti inflammatory activity can be determined by using variousexperimental and spontaneous animal models of inflammatory arthritisknown in the art and described in Crofford L. J. and Wilder R. L.,“Arthritis and Autoimmunity in Animals”, in Arthritis and AlliedConditions: A Textbook of Rheumatology, McCarty et al. (eds.), Chapter30 (Lee and Febiger, 1993). For example, adjuvant-induced arthritismodels such as carrageenan-, xymosan-, or collagen-induced arthritis inrats, hamsters, rabbits, dogs and pigs, are useful in studyinganti-inflammatory activity, and inhibition of carrageenan-induced pawedema in rats is a primary in vivo screen for the anti inflammatoryactivity of most NSAIDs, and is considered predictive of human efficacy.These models are described in, e.g., Winter et al. (1962) Proc. Soc.Exp. Biol Med. 111:544-47; and Hansra et al. (2000) Inflammation24(2):141-55. Animal models for inflammatory bowel disease can also beused to assess the efficacy of therapies of the invention, for examplethe models described in, e.g., Strober (1985) Dig. Dis. Sci. 30(12Suppl):3S-10S; Kim et al. (1992) Scand. J. Gastroentrol. 27:529-37). Inthese models, ulcerative cholitis and Crohn's disease can be induced inanimals by oral administration of sulfated polysaccharides, dextransulfate or chemical irritants.

Efficacy in treating autoimmune disorders may be assessed using animalmodels for autoimmune disorders such as type 1 diabetes, thyroidautoimmunity, systemic lupus eruthematosus, and glomerulonephritis, forexample the models described in Flanders et al. (1999) Autoimmunity29:235-46; Krogh et al. (1999) Biochimie 81:511-15; Foster (1999) Semin.Nephrol. 19:12-24, etc.

The anti-cancer activity of the therapeutic agents also can bedetermined by using various experimental animal models for the study ofcancer such as the SCID mouse model, transgenic mice or nude mice withhuman xenografts, and other animal models such as hamsters, rabbits,etc. known in the art and described in Relevance of Tumor Models forAnticancer Drug Development (1999, eds. Fiebig and Burger);Contributions to Oncology (1999, Karger); The Nude Mouse in OncologyResearch (1991, eds. Boven and Winograd); and Anticancer DrugDevelopment Guide (1997 ed. Teicher). Preferred animal models are mousexenograft models. Tumor cell lines that can be used as a source forxenograft tumors include but are not limited to, SKBR3 and MCF7 cells,which can be derived from patients with breast adenocarcinoma. Thesecells have both erbB2 and prolactin receptors. SKBR3 cells have beenused routinely in the art as ADCC and xenograft tumor models.Alternatively, OVCAR3 cells derived from a human ovarian adenocarcinomacan be used as a source for xenograft tumors.

The therapeutic agents of the invention are preferably tested in vitro,and then in vivo, for the desired therapeutic or prophylactic activity,prior to use in humans. Therapeutic agents and methods may be screenedusing cells of a tumor or malignant cell line. Many assays standard inthe art can be used to assess such survival and/or growth; for example,cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the therapeutic agents for usein humans. The dosage of such agents lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyagent used in the method of the invention, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

D. Other Methods D1. Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingmolecules of the invention, are administered to treat, prevent orameliorate one or more symptoms associated with a disease, disorder, orinfection, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded antibody or fusion protein thatmediates a therapeutic or prophylactic effect. Any methods for genetherapy available in the art may be used, for example the methodsdescribed in, e.g., Goldspiel et al. (1993) Clinical Pharmacy12:488-505; Wu and Wu (1991) Biotherapy 3:87-95; Tolstoshev (1993) Ann.Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993) Science260:926-932; and Morgan and Anderson (1993) Ann. Rev. Biochem.62:191-217.

In a preferred aspect, a composition of the invention comprises nucleicacids encoding an antibody, diabody, or fusion protein of the invention,said nucleic acids being part of an expression vector that expresses theantibody in a suitable host. In particular, such nucleic acids havepromoters, preferably heterologous promoters, operably linked to theantibody coding region, said promoter being inducible or constitutive,and, optionally, tissue-specific. In another particular embodiment,nucleic acid molecules are used in which the antibody coding sequencesand any other desired sequences are flanked by regions that promotehomologous recombination at a desired site in the genome, thus providingfor intrachromosomal expression of the antibody encoding nucleic acids,as described in Koller and Smithies (1989) Proc. Natl. Acad. Sci.(U.S.A.) 86:8932-35; and Zijlstra et al. (1989) Nature 342:435-38.

Delivery of the nucleic acids into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, a polynucleotide encoding a polypeptide of thepresent invention is administered in vivo, where it is expressed toproduce the encoded polypeptide. This can be accomplished by any ofnumerous methods, such as by infection using retroviral or other viralvectors (as described in, e.g., U.S. Pat. No. 4,980,286; Miller et al.(1993) Meth. Enzymol. 217:581-599; Salmons and Gunzberg (1993) HumanGene Therapy 4:129-141; Grossman and Wilson (1993) Curr. Opin. inGenetics and Devel. 3:110-114; Kozarsky and Wilson (1993) Current Op. inGenetics and Dev. 3:499-503; Walsh et al. (1993) Proc. Soc. Exp. Biol.Med. 204:289-300; Bout et al. (1994) Human Gene Therapy 5:3-10; Boesenet al. (1994) Biotherapy 6:291-302; Clowes et al. (1994) J. Clin.Invest. 93:644-651; Klein et al. (1994) Blood 83:1467-1473; and U.S.Pat. No. 5,436,146), or by direct injection of naked DNA, or by use ofmicroparticle bombardment (e.g., a gene gun), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus or in linkageto an antigen subject to receptor-mediated endocytosis (as described in,e.g., Wu and Wu (1987) J. Biol. Chem. 262:4429-4432; Joliot et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:1864-1868; WO 92/06180; WO92/22635; W092/20316; W093/14188; WO 93/20221) (which can be used totarget cell types specifically expressing the receptors), etc.

A nucleic acid may be introduced into a cell prior to administration invivo of the resulting recombinant cell, for example as described in WO94/08598; Rheinwald (1980) Meth. Cell Bio. 21A:229; Pittelkow and Scott(1986) Mayo Clinic Proc. 61:771; Stemple and Anderson (1992) Cell 71:973-985. The resulting recombinant cells can be delivered to a subjectby various methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art. Cells into which a nucleic acid can be introducedfor purposes of gene therapy encompass any desired, available cell type,and include but are not limited to epithelial cells, endothelial cells,keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells suchas T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc. In a preferred embodiment, the cell used for gene therapy isautologous to the subject.

D2. Vaccine Therapy

In some embodiments, the antibodies of the invention may be used toinduce an immune response against an antigenic or immunogenic agent,including but not limited to cancer antigens and infectious diseaseantigens. The vaccine compositions of the invention comprise one or moreantigenic or immunogenic agents to which an immune response is desired,wherein the one or more antigenic or immunogenic agents is coated withan antibody of the invention. The vaccine compositions of the inventionare particularly effective in eliciting an immune response, preferably aprotective immune response against the antigenic or immunogenic agent,which may be a virus against which an immune response is desired, or anantigen derived from other viral or non-viral pathogens.

In yet other embodiments, the invention encompasses pathogenic cells orviruses, preferably attenuated viruses, which express the antibody ontheir surface. The invention further encompasses methods to inducetolerance in a subject by administering a composition of the invention.Preferably a composition suitable for inducing tolerance in a subject,comprises an antigenic or immunogenic agent coated with an antibody ofthe invention.

D3. Targeting Liposomes or Other Microcarriers and Nanocarriers

In some embodiments, the antibodies of the invention can be used toprepare targeted liposomes for delivery of a desired therapeuticcomposition (e.g., anti-cancer agents) to a target cell (e.g., aprostate cancer cell or other HER2/neu expressing cell). The preparationand use of immunoliposomes for targeted delivery of antitumor drugs isreviewed in Mastrobattista et al. (1999) Advanced Drug Delivery Reviews40:103-127. Liposomes are vesicular structures based on lipid bilayers.They can be as small as 20 nm and as large as 10 μm in diameter. Theycan be unilamellar (only one bilayer surrounds an aqueous core) ormultilamellar (two or more bilayers concentrically oriented around anaqueous core). Targeting of liposomes using a variety of targetingagents (e.g., antibodies of the invention) is well known in the art.See, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044. Standard methods forcoupling targeting agents to liposomes can be used. Antibody targetedliposomes can be constructed using, for instance, liposomes whichincorporate protein A. See Renneisen et al. (1990) J. Biol. Chem.265:16337-16342; and Leonetti et al. (1990) Proc. Natl. Acad. Sci.(U.S.A.) 87:2448-2451.

In a preferred embodiment, the liposomes are formed from standardvesicle-forming lipids, which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of, e.g., liposome sizeand stability of the liposomes in the bloodstream. A variety of methodsare available for preparing liposomes, as described in, e.g., Szoka, etal. (1980) Ann. Rev. Biophys. Bioeng. 9:467; U.S. Pat. Nos. 4,235,871;4,501,728; and 4,837,028. One method produces multilamellar vesicles ofheterogeneous sizes. In this method, the vesicle forming lipids aredissolved in a suitable organic solvent or solvent system and driedunder vacuum or an inert gas to form a thin lipid film. If desired, thefilm may be redissolved in a suitable solvent, such as tertiary butanol,and then lyophilized to form a more homogeneous lipid mixture which isin a more easily hydrated powder-like form. This film is covered with anaqueous solution of the targeted drug and the targeting component(antibody) and allowed to hydrate, typically over a 15-60 minute periodwith agitation. The size distribution of the resulting multilamellarvesicles can be shifted toward smaller sizes by hydrating the lipidsunder more vigorous agitation conditions or by adding solubilizingdetergents such as deoxycholate.

D4. Immunoassays

The antibodies of the invention can be used to detect HER2/neu, or cellsexpressing HER2/neu. Any of a number of methods may be used to achievesuch detection. For example, immunological binding assays may be used(see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and4,837,168). For a review of the general immunoassays, see also Asai (ed.1993) Methods in Cell Biology Vol. 37, Academic Press, New York; Stites& Terr (eds. 1991) Basic and Clinical Immunology 7th Ed.

Thus, the present invention provides methods of detecting cells thatexpress HER2/neu. In one method, a biopsy is performed on the subjectand the collected tissue is tested in vitro. The tissue or cells fromthe tissue is then contacted, with an anti-HER2/neu antibody of theinvention. Any immune complexes which result indicate the presence of aHER2/neu protein in the biopsied sample. To facilitate such detection,the antibody can be radiolabeled or coupled to an effector moleculewhich is a detectable label, such as a radiolabel. In another method,the cells can be detected in vivo using typical imaging systems. Then,the localization of the label is determined by any of the known methodsfor detecting the label. A conventional method for visualizingdiagnostic imaging can be used. For example, paramagnetic isotopes canbe used for MRI. Internalization of the antibody may be important toextend the life within the organism beyond that provided byextracellular binding, which will be susceptible to clearance by theextracellular enzymatic environment coupled with circulatory clearance.

HER2/neu proteins can also be detected using standard immunoassaymethods and the antibodies of the invention. Standard methods include,for example, radioimmunoassay, immunochromatographic methods, sandwichimmunoassays (including ELISA), immunofluorescence assays, Western blot,affinity chromatography (affinity ligand bound to a solid phase), and insitu detection with labeled antibodies.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention unless specified.

EXAMPLE 1 BIACore Affinity Determinations

The kinetic parameters of the binding of eluted and purified antibodieswere analyzed using a BIAcore assay (BIAcore instrument 1000, BIAcoreInc., Piscataway, N.J.) and associated software. HER-2 was immobilizedon one of the four flow cells (flow cell 2) of a sensor chip surfacethrough amine coupling chemistry (by modification of carboxymethylgroups with mixture of NHS/EDC) such that about 1000 response units (RU)of receptor was immobilized on the surface. Following this, theunreacted active esters were “capped off” with an injection of 1MEt-NH2. Once a suitable surface was prepared, ch4D5-FcWT (wild-type Fc),ch4D5, and trastuzumab (control) were injected at concentrations of6.25-200 nM over the surface at a flow rate of 70 mL/min for 180 sec.

Once an entire data set was collected, the resulting binding curves wereglobally fitted and the rate constants and apparent equilibrium bindingconstant were calculated using computer algorithms supplied by themanufacturer, as described in the BIAevaluation Software Handbookavailable from BIAcore, Inc. FIG. 3 shows the graphical results of theSPR analysis, and the calculated constants are provided in Table 5.

TABLE 5 Kinetic and Equilibrium Constants Calculated from BIAcore DataAnalyte Ka1 (1/mole * s) Kd1 (1/s) K_(D) (nm) ch4D5-wild-type Fc 1.7 ×10⁵ ~3.2 × 10⁻⁷ (est.) — ch4D5 1.1 × 10⁵ ~6.3 × 10⁻⁶ (est.) —trastuzumab 1.6 × 10⁵   1.3 × 10⁻⁴ 0.8

EXAMPLE 2 Apoptosis

Various cell lines were incubated overnight with ch4D5 and ch4D5-FcMT1.Apoptosis was assayed by FACS analysis, and results are shown in Table6.

EXAMPLE 3 Proliferation

[³H]Thymidine ([³H]TdR) incorporation into DNA was used as a biochemicalindex of SKBR3 cell proliferation, to compare the effects of variouschimeric 4D5 antibodies of the present embodiments. The effect ofch4D5-Ag, ch4D5, and Ch4D-FcMT1 on CD16-158F+ and CD16-158V+ cells werestudied and compared to controls. Results are depicted in FIG. 4.

EXAMPLE 4 Anti-Tumor Activity in Mice (Breast Cancer Model)

Anti-tumor activity of various antibodies was studied in a breast cancermodel using non-transgenic and transgenic (hCD16A) mice. Fifty Balb/cRAG2−/− non-transgenic mice from MacroGenics breeding colony wereinjected s.c. at day 0 with JMT-1 breast cancer cells. Mice were dividedinto five groups of 10 mice each, and treated intraperitoneously (IP)weekly for 8 weeks with ch4D5 N297Q, ch4D5-wild-type Fc, ch4D5-FcMT1,ch4D5-FcMT2, or PBS (negative control). Tumor development is monitoredtwice per week, using calipers, and tumor weight is estimated by thefollowing formula: tumor weight=(length×width²)/2. Results are shown inFIG. 5. Twenty-three Balb/c RAG2−/− mCD16−/− hCD16A+ transgenic micefrom MacroGenics breeding colony were injected s.c. at day 0 with JIMT-1breast cancer cells. Mice were divided into three groups, and treatedintraperitoneously (IP) weekly for 8 weeks with ch4D5-wild-type Fc(n=8), ch4D5-FcMT1 (n=8), or PBS (negative control; n=7). Tumordevelopment is monitored twice per week, using calipers, and tumorweight is estimated by the following formula: tumorweight=(length×width²)/2. Results are shown in FIG. 6.

EXAMPLE 5 Anti-Tumor Activity in Mice (Ovarian Cancer Model)

Anti-tumor activity of various antibodies was studied in an ovariancancer model using non-transgenic and transgenic (hCD16A) mice. 22 R3−/−N/N non-transgenic mice from MacroGenics breeding colony were injecteds.c. at day 0 with SKOV-3 ovarian cancer cells. Mice were divided intofour groups, and treated intraperitoneously (IP) weekly for 8 weeks withch4D5 N297Q (n=5), ch4D5-wild-type Fc (n=6), ch4D5-FcMT1 (n=6), or PBS(negative control; n=5). Tumor development is monitored twice per week,using calipers, and tumor weight is estimated by the following formula:tumor weight=(length×width²)/2. Results are shown in FIG. 7, Panel A. 32R3−/− N/N hCD16A+ transgenic mice from MacroGenics breeding colony wereinjected s.c. at day 0 with SKOV-3 ovarian cancer cells. Mice weredivided into four groups, and treated intraperitoneously (IP) weekly for8 weeks with ch4D5 N297Q (n=8), ch4D5-wild-type Fc (n=8), ch4D5-FcMT1(n=8), or PBS (negative control; n=8). Tumor development is monitoredtwice per week, using calipers, and tumor weight is estimated by thefollowing formula: tumor weight=(length×width²)/2. Results are shown inFIG. 7, Panel B. 96 mCD16−/− huCD16A FoxN1−/− (nu/nu) transgenic micefrom MacroGenics breeding colony were injected s.c. at day 0 with SKOV-3ovarian cancer cells. Mice were divided into six groups of 16 mice each,and treated intraperitoneously (IP) weekly for 8 weeks with ch4D5-FcMT3,ch4D5-FcMT1, ch4D5-FcMT4, ch4D5, ch4D5Ag, or PBS (negative control).Tumor development is monitored twice per week, using calipers, and tumorweight is estimated by the following formula: tumorweight=(length×width²)/2. Results are shown in FIG. 8.

EXAMPLE 6 ADCC Assays in Various Cancer Cell Lines

FIG. 9 illustrates representative immunohistochemical staining ofvarious cancer cell lines for HER2/neu. Cell lines were ranked accordingto their HER2/neu staining intensity as specified in the HER2/neu testkit sold as DAKO HerceptTest™ (DakoCytomation, Glostrup, Denmark):missing HER2/neu staining (DAKO score 0); weak HER2/neu staining (DAKOscore 1+); moderate HER2/neu staining (DAKO score 2+); and strongHER2/neu staining (DAKO score 3+). The various panels represent thevarious cell lines, as shown in Table 7.

TABLE 7 DAKO Staining of Various Cancer Cell Lines in FIG. 9 Panel CellLine Description Sites/Cell Score A MDA-MB-435 Breast carcinoma 4.7 ×10³ 0 B MDA-MB-231 Breast adenocarcinoma 1.6 × 10⁴ 0 C A549 Lungadenocarcinoma 3.4 × 10⁴ 1+ D OVCAR-8 Ovarian carcinoma 4.4 × 10⁴ 1+ EMCF-7 Breast adenocarcinoma 4.5 × 10⁴ 1+ F BT-20 Ductal carcinoma 6.9 ×10⁴ 1+ G HT-29 Colon/Colorectal cancer 9.4 × 10⁴ 1+ H ZR75-1 Ductalcarcinoma 1.4 × 10⁵ 2+ I JIMT-1 Breast carcinoma 2.0 × 10⁵ 2+ JMDA-MB-453 Breast carcinoma 2.8 × 10⁵ 3+ K BT-474 Ductal carcinoma 2.0 ×10⁶ 3+ L SKBR-3 Breast carcinoma 3.0 × 10⁶ 3+ M mSKOV-3 Ovarian cancer4.0 × 10⁶ 3+

Several ch4D5 antibodies including ch4D5 antibodies having Fc variantdomains were tested for the ability to mediate ADCC in the cancer celllines, including ch4D5-FcMT1, ch4D5-FcMT2, ch4D5-FcMT3, ch4D5-FcWT(wild-type Fc), ch4D5 N297Q and trastuzumab (as a control). Data fromvalid assays (SR≦20% MR, AICC≦50% MR) is reported in Table 8, where EC₅₀estimates were considered valid only if the model fit a max lysisof >20%. Comparison of EC₅₀ and max lysis parameters was performed byasking whether the best fit values obtained for the Fc-optimizedantibodies were statistically different from those obtained for the Fcwild-type ch4D5 antibody by the sum-of-squares F test. Data were alsofitted to sigmoidal dose-response models as shown in FIGS. 10-13.

TABLE 8 ADCC Assays in Various Cell Lines Max EC50 Lysis FIG. Cell LineAntibody (ng/mL) p (%) p (Panel) MDA-MB- ch4D5-FcMT1 ND — 5 NS 10 (A)435 ch4D5-FcMT2 ND — 13 NS ch4D5-FcMT3 ND — 7 NS ch4D5-FcWT ND — 7 —trastuzumab ND — 7 NS MDA-MB- ch4D5-FcMT1 4 NS 27 NS 10 (B) 231ch4D5-FcMT2 12 NS 29 NS ch4D5-FcMT3 ? ? 24 NS ch4D5-FcWT 9 — 27 —trastuzumab 7 NS 22 NS A549 ch4D5-FcMT1 14 — 34 <0.01 11 (A) ch4D5-FcMT221 — 24 <0.01 ch4D5-FcMT3 >100 — 23 <0.01 ch4D5-FcWT ND — 6 —trastuzumab ND — 5 NS OVCAR-8 ch4D5-FcMT1 14 <0.01 43 <0.01 11 (B)ch4D5-FcMT2 21 <0.05 40 <0.01 ch4D5-FcMT3 26 NS 36 <0.01 ch4D5-FcWT 57 —16 — trastuzumab 37 NS 13 NS MCF-7 ch4D5-FcMT1 4 <0.05 55 <0.01 11 (C)ch4D5-FcMT2 9 NS 51 <0.01 ch4D5-FcMT3 8 NS 48 <0.01 ch4D5-FcWT 23 NS 32— trastuzumab 9 — 21 NS BT-20 ch4D5-FcMT1 42 <0.01 66 <0.01 11 (D)ch4D5-FcMT2 78 <0.01 62 <0.01 ch4D5-FcMT3 67 <0.01 55 <0.01ch4D5-FcWT >100 — 33 — trastuzumab >100 NS 25 NS HT-29 ch4D5-FcMT1 0.4 —43 <0.01 11 (E) ch4D5-FcMT2 0.5 — 44 <0.01 ch4D5-FcMT3 1 — 38 <0.01ch4D5-FcWT ND — 13 — ZR75-1 ch4D5-FcMT1 14 <0.01 78 <0.01 12 (A)ch4D5-FcMT2 20 NS 67 <0.01 ch4D5-FcMT3 26 <0.01 63 <0.01 ch4D5-FcWT 38 —38 — trastuzumab ND — 23 <0.01 JIMT-1 ch4D5-FcMT1 8 NS 73 <0.01 12 (B)ch4D5-FcMT2 7 <0.05 70 <0.01 ch4D5-FcMT3 10 NS 65 <0.01 ch4D5-FcWT 22 —43 — trastuzumab 10 NS 34 NS MDA-MB- ch4D5-FcMT1 3 <0.05 59 <0.01 13 (A)453 ch4D5-FcMT2 4 <0.05 58 <0.01 ch4D5-FcMT3 6 NS 57 <0.01 ch4D5-FcWT 11— 45 — trastuzumab 3 <0.05 31 <0.01 BT-474 ch4D5-FcMT1 3 <0.01 73 <0.0113 (B) ch4D5-FcMT2 3 <0.05 58 NS ch4D5-FcMT3 4 <0.05 71 NS ch4D5-FcWT 11— 64 — trastuzumab 7 NS 60 NS SKBR-3 ch4D5-FcMT1 0.4 <0.01 64 NS 13 (C)ch4D5-FcMT3 0.8 <0.01 61 NS ch4D5-FcWT 6 — 62 — mSKOV-3 ch4D5-FcMT1 1.2NS 71 <0.01 13 (D) ch4D5-FcMT2 7 <0.05 43 <0.05 ch4D5-FcMT3 0.9 <0.05 56NS ch4D5-FcWT 3 — 58 —

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

1-20. (canceled)
 21. A method of treating a HER2/neu-expressing cancerin a patient having a cancer, comprising administering to said patient atherapeutically effective amount of an antibody that binds humanHER2/neu and comprises: (A) an immunoglobulin light chain comprising anamino acid sequence of SEQ ID NO: 2; and (B) an immunoglobulin heavychain comprising an amino acid sequence of SEQ ID NO:
 11. 22. The methodof claim 21, wherein said antibody is administered by intravenousinfusion.
 23. The method of claim 21, comprising the further step ofadministering a second therapeutic agent simultaneously or sequentiallywith said antibody.
 24. The method of claim 23, wherein said secondtherapeutic agent and said antibody are administered no more than 12hours apart, or no more than 24 hours apart, or no more than 96 hoursapart.
 25. The method of claim 23, wherein said second therapeutic agentis selected from the group consisting of an anti-angiogenic agent, ananti-neoplastic agent, a chemotherapeutic agent, and a cytotoxic agent.26. The method of claim 23, wherein said second therapeutic agent isselected from the group consisting of an anti-mitotic agent, a hormonetherapy agent, and an aromatase inhibitor.
 27. A pharmaceuticalcomposition comprising an antibody that binds human HER2/neu andcomprises: (A) an immunoglobulin light chain comprising an amino acidsequence of SEQ ID NO: 2; and (B) an immunoglobulin heavy chaincomprising an amino acid sequence of SEQ ID NO: 11; and (C) apharmaceutically acceptable carrier and/or an excipient.
 28. Thepharmaceutical composition of claim 27, wherein said pharmaceuticalcomposition is a sterile lyophilized powder in a hermetically sealedcontainer.
 29. The pharmaceutical composition of claim 28, wherein saidsterile lyophilized powder comprises least 5 mg, at least 10 mg, atleast 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50mg, or at least 75 mg of said antibody.
 30. The pharmaceuticalcomposition of claim 27, wherein said pharmaceutical composition is asterile liquid in a hermetically sealed container.
 31. Thepharmaceutical composition of claim 30, wherein the concentration ofsaid antibody in said sterile liquid is at least 1 mg/ml, at least 2.5mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, atleast 150 mg/ml, or at least 200 mg/ml.
 32. A kit comprising thepharmaceutical composition of claim 27 and instructions foradministration.
 33. A kit comprising the pharmaceutical composition ofclaim 28 and instructions for administration.
 34. A kit comprising thepharmaceutical composition of claim 30 and instructions foradministration.
 35. A method of treating a HER2/neu-expressing cancer ina patient having a cancer, comprising administering to said patient atherapeutically effective amount of the pharmaceutical composition ofclaim
 27. 36. A method of treating a HER2/neu-expressing cancer in apatient having a cancer, comprising administering to said patient atherapeutically effective amount of the pharmaceutical composition ofclaim
 28. 37. A method of treating a HER2/neu-expressing cancer in apatient having a cancer, comprising administering to said patient atherapeutically effective amount of the pharmaceutical composition ofclaim
 30. 38. An antibody that binds human HER2/neu and comprises: (A)an immunoglobulin light chain comprising an amino acid sequence of SEQID NO: 2; and (B) an immunoglobulin heavy chain comprising an amino acidsequence of SEQ ID NO:
 11. 39. A method of treating aHER2/neu-expressing cancer in a patient having a cancer, comprisingadministering to said patient a therapeutically effective amount of saidantibody of claim
 38. 40. A pharmaceutical composition comprising saidantibody of claim 38, and a pharmaceutically acceptable carrier and/oran excipient.